U.S. patent number 9,956,264 [Application Number 15/000,637] was granted by the patent office on 2018-05-01 for mic-1 fusion proteins and uses thereof.
This patent grant is currently assigned to Novo Nordisk A/S. The grantee listed for this patent is Novo Nordisk A/S. Invention is credited to Kim V. Andersen, Sven Hastrup, Charlotte Helgstrand, Sebastian B. Joergensen, Michael P. Bastner Sandrini, Kristian Sass-Oerum, Allan C. Shaw, Henning Thoegersen.
United States Patent |
9,956,264 |
Shaw , et al. |
May 1, 2018 |
MIC-1 fusion proteins and uses thereof
Abstract
The invention relates to MIC-1 fusion proteins. More
specifically it relates to compounds comprising fusion proteins
comprising a MIC-1 protein or an analog thereof at the C-terminus
of the fusion protein and a functional variant of human serum
albumin at the N-terminus of the fusion protein connected via a
peptide linker. The compounds of the invention have MIC-1 activity.
The invention also relates to pharmaceutical compositions
comprising such compounds and pharmaceutically acceptable
excipients, as well as the medical use of the compounds.
Inventors: |
Shaw; Allan C. (Copenhagen N,
DK), Helgstrand; Charlotte (Bagsvaerd, DK),
Sandrini; Michael P. Bastner (Bagsvaerd, DK),
Joergensen; Sebastian B. (Bagsvaerd, DK), Thoegersen;
Henning (Farum, DK), Sass-Oerum; Kristian
(Koebenhavn, DK), Hastrup; Sven (Koebenhavn,
DK), Andersen; Kim V. (Bagsvaerd, DK) |
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
N/A |
DK |
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Assignee: |
Novo Nordisk A/S (Bagsvaerd,
DK)
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Family
ID: |
51032955 |
Appl.
No.: |
15/000,637 |
Filed: |
January 19, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160129083 A1 |
May 12, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14742159 |
Jun 17, 2015 |
9272019 |
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Foreign Application Priority Data
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Jun 24, 2014 [EP] |
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14173664 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/475 (20130101); A61K 38/18 (20130101); C07K
14/765 (20130101); A61K 38/385 (20130101); A61K
38/19 (20130101); C07K 14/00 (20130101); C07K
14/52 (20130101); A61K 47/65 (20170801); A61P
3/04 (20180101); A61K 47/643 (20170801); C07K
2319/31 (20130101) |
Current International
Class: |
A61K
38/18 (20060101); A61K 38/19 (20060101); A61K
47/64 (20170101); C07K 14/475 (20060101); C07K
14/00 (20060101); A61K 47/65 (20170101); C07K
14/765 (20060101); C07K 14/52 (20060101); A61K
38/38 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0179271 |
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Oct 2001 |
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WO |
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0179443 |
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Oct 2001 |
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WO |
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2005099746 |
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Oct 2005 |
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WO |
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2009/023270 |
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Feb 2009 |
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WO |
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2013113008 |
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Aug 2013 |
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WO |
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2013148117 |
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Oct 2013 |
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WO |
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2014120619 |
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Aug 2014 |
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WO |
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2015/017710 |
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Feb 2015 |
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WO |
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Other References
Arai R. et al., Design of the linkers which effectively separate
domains of a bifunctional fusion protein, Protein Engineering,
2001, vol. 14, No. 8, pp. 529-532. cited by applicant .
Bauskin A. R. et al., The propeptide of macrophage inhibitory
cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality
control determinant for correctly folded MIC-1, The EMBO Journal,
2000, vol. 19, No. 10, pp. 2212-2220. cited by applicant .
Bootcov M. R. et al., MIC-1, a novel macrophage inhibitory
cytokine, is a divergent member of the TGF-b superfamily,
Proceedings of the National Academy of Sciences, 1997, vol. 94, pp.
11514-11519. cited by applicant .
Chen X. et al., Fusion protein linkers: property, design and
functionality, Advanced Drug Delivery Reviews, 2012, vol. 65, No.
10, pp. 1357-1369. cited by applicant .
Lu Z. et al., Change of body weight and macrophage inhibitory
cytokine-1 during chemotherapy in advanced gastric cancer: what is
their clinical significance?, Public Library of Science One, 2014,
vol. 9, No. 2, p. e88553. cited by applicant .
Robinson et al "Structure-dependent nonenzymatic deamidation of
glutaminyl and asparaginyl pentapeptides" J. Peptide Res 2004 vol.
63 No. 5 pp. 426-436. cited by applicant.
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Primary Examiner: Bradley; Christina
Attorney, Agent or Firm: Lum; Leon Y.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
14/742,159 filed Jun. 17, 2015 which claims priority under 35
U.S.C. .sctn. 119 to European Patent Application 14173664.5, filed
Jun. 24, 2014; the contents of which is incorporated herein by
reference.
Claims
The invention claimed is:
1. A fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin or a functional variant thereof; B is a peptide
linker 10 to 50 amino acids in length and comprising the formula
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is from 2 to 10; and C is a MIC-1 protein or an
analogue thereof; wherein the C-terminus of the human serum albumin
or functional variant thereof is fused to the N-terminus of the
peptide linker; and wherein the C-terminus of the peptide linker is
fused to the N-terminus of the MIC-1 protein or analogue
thereof.
2. A compound comprising a homodimer of a fusion protein of formula
(I): A-B-C (I), wherein A is human serum albumin or a functional
variant thereof; B is a peptide linker 10 to 50 amino acids in
length and comprising the formula [X-Y.sub.m].sub.n, wherein X is
Asp or Glu; Y is Ala; m is from 2 to 4; and n is from 2 to 10; and
C is a MIC-1 protein or an analogue thereof; wherein the C-terminus
of the human serum albumin or functional variant thereof is fused
to the N-terminus of the peptide linker; wherein the C-terminus of
the peptide linker is fused to the N-terminus of the MIC-1 protein
or analogue thereof; and wherein the homodimer comprises an
interchain disulphide bridge between the MIC-1 protein or analogue
thereof of each fusion protein.
3. A pharmaceutical composition comprising a fusion protein of
formula (I): A-B-C (I), wherein A is human serum albumin or a
functional variant thereof; B is a peptide linker 10 to 50 amino
acids in length and comprising the formula [X-Y.sub.m].sub.n,
wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is from
2 to 10; and C is a MIC-1 protein or an analogue thereof; wherein
the C-terminus of the human serum albumin or functional variant
thereof is fused to the N-terminus of the peptide linker; and
wherein the C-terminus of the peptide linker is fused to the
N-terminus of the MIC-1 protein or analogue thereof; or a
pharmaceutically acceptable salt, amide, or ester thereof.
4. The pharmaceutical composition according to claim 3, further
comprising a homodimer of the fusion protein, wherein the homodimer
comprises an interchain disulphide bridge between the MIC-1 analog
of each fusion protein.
5. A method of treating obesity by administering a pharmaceutically
active amount of a fusion protein of formula (I): A-B-C (I),
wherein A is human serum albumin or a functional variant thereof; B
is a peptide linker 10 to 50 amino acids in length and comprising
the formula [X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m
is from 2 to 4; and n is from 2 to 10; and C is a MIC-1 protein or
an analogue thereof; wherein the C-terminus of the human serum
albumin or functional variant thereof is fused to the N-terminus of
the peptide linker; and wherein the C-terminus of the peptide
linker is fused to the N-terminus of the MIC-1 protein or analogue
thereof.
6. The method according to claim 5, further comprising
administering a homodimer of the fusion protein, wherein the
homodimer comprises an interchain disulphide bridge between the
MIC-1 analog of each fusion protein.
7. The fusion protein according to claim 1, wherein A is selected
from the group consisting of SEQ ID NO:2 and SEQ ID NO:23.
8. The fusion protein according to claim 1, wherein B is selected
from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID
NO:38.
9. The fusion protein according to claim 1, wherein C is selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and
SEQ ID NO:22.
10. The fusion protein according to claim 1, wherein A is selected
from the group consisting of SEQ ID NO:2 and SEQ ID NO:23; wherein
B is selected from the group consisting of SEQ ID NO:4, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,
and SEQ ID NO:38; and wherein C is selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.
Description
TECHNICAL FIELD
The present invention relates to MIC-1 fusion proteins and their
pharmaceutical use.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Jan. 13,
2016, is named 140026US01_SL_ST25.txt and is 28,566 bytes in
size.
BACKGROUND
The macrophage inhibitory cytokine-1 (MIC-1), also known as GDF-15
and placental bone morphogenetic protein (PLAB), is a distant
member of the TGF-beta super family, a family of peptide hormones
involved in cell growth and differentiation. MIC-1 circulates as a
cysteine-rich homodimer with a molecular mass of 24.5 kDa. MIC-1
was initially reported to be up-regulated in macrophages by stimuli
including IL-1b, TNF-alpha, IL-2, and TGF-b. It was also shown that
MIC-1 could reduce lipopolysaccharide-induced TNF-alpha production
and it was based on these data proposed that MIC-1 was an
anti-inflammatory cytokine. More recently, a study was
investigating why human patients with advanced cancer were losing
body weight and they showed that the weight loss correlated with
circulating levels of MIC-1. These data indicates that MIC-1
regulates body weight. This hypothesis was tested in mice
xenografted with prostate tumor cells, where data showed that
elevated MIC-1 levels were associated with loss of body weight and
decreased food intake, this effect being reversed by administration
of antibodies to MIC-1. As administration of recombinant MIC-1 to
mice regulated hypothalamic neuropeptide Y and pro-opiomelanocortin
it was proposed that MIC-1 regulates food intake by a central
mechanism. Furthermore, transgenic mice overexpressing MIC-1 are
gaining less weight and body fat both on a normal low fat diet and
on a high fat diet. Also, transgenic mice overexpressing MIC-1 fed
both on a low and high fat diet, respectively, had improved glucose
tolerance compared with wild type animals on a comparable diet.
Native MIC-1 has a short half-life, meaning that treatment with
native MIC-1 requires daily administration to maintain
efficacy.
WO 2001079443 concerns the use of human serum albumin or variants
thereof for fusions to peptides of pharmaceutical interest.
WO 2005099746 concerns a method of modulating appetite and/or body
weight by administering a MIC-1 modulating agent.
SUMMARY
The invention relates to MIC-1 fusion proteins.
In one aspect, the invention provides compounds comprising fusion
proteins comprising a MIC-1 protein or an analogue thereof at the
C-terminus of the fusion protein and a functional variant of human
serum albumin (HSA) at the N-terminus of the fusion protein
connected via a peptide linker. The peptide linker has a length of
10 to 50 amino acids and comprises the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4, and n is at least 2.
In one aspect of the invention, Y is selected from the group of
coded amino acids except for Pro and Gly. In another aspect, Y is
selected from the group of coded non-polar amino acids, except for
Pro and Gly.
In one aspect, the invention provides a polynucleotide molecule
encoding a compound comprising a fusion protein comprising a MIC-1
protein or an analogue thereof at the C-terminus of the fusion
protein and a functional variant of HUMAN SERUM ALBUMIN at the
N-terminus of the fusion protein connected via a peptide
linker.
In one aspect, the invention provides a pharmaceutical composition
comprising a compound of the invention or a pharmaceutically
acceptable salt, amide or ester thereof, and one or more
pharmaceutically acceptable excipients.
In one aspect, the invention provides a compound of the invention
for use as a medicament.
In one aspect, the invention provides a compound of the invention
for use in the treatment of eating disorders, such as obesity, e.g.
by decreasing food intake, reducing body weight, suppressing
appetite and inducing satiety.
In one aspect, the invention provides a compound of the invention
for use in the treatment of obesity.
In one aspect, the compounds of the invention are MIC-1 agonists.
In one aspect, the compounds of the invention inhibit food intake.
In one aspect, the compounds of the invention reduce body
weight.
In one aspect, the compounds of the invention have longer half-life
than the half-life of native MIC-1.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1. Schematic representation of a HSA-MIC-1 dimeric fusion
protein. A, B and C depicts relative positions of the human serum
albumin domain, the linker region and MIC-1, respectively. --SS--
indicates interchain disulphide bridge linking together the two
HSA-MIC-1 monomers to form a functional dimeric fusion protein.
DESCRIPTION
The invention relates to compounds comprising MIC-1 fusion
proteins. In one aspect, the invention relates to MIC-1 fusion
proteins.
In one aspect, the invention provides compounds comprising fusion
proteins comprising MIC-1 or an analogue thereof at the C-terminus
of the fusion protein and human serum albumin (HSA) or a functional
variant thereof at the N-terminus of the fusion protein connected
via a peptide linker. The peptide linker has a length of 10 to 50
amino acids and comprises the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4, and n is at least 2.
In one aspect of the invention, Y is selected from the group of
coded amino acids except for Pro and Gly. In another aspect, Y is
selected from the group of coded non-polar amino acids, except for
Pro and Gly.
The fusion protein strategy of the present invention combines the
soluble, stable plasma protein human serum albumin with native
MIC-1 or a MIC-1 analogue. Human serum albumin has inherent
properties such as high solubility and stability which makes it
beneficial to use as fusion partner for improving expression yield
and conferring stability to MIC-1. Human serum albumin as fusion
partner may also increase the plasma half-life of MIC-1 by
significant size increase, which inhibits renal clearance and/or by
binding the Fc Neonatal Receptor, which allows recycling from the
endosome and prevention of lysomal degradation allowing the
molecule to be present longer in circulation. As with other smaller
therapeutic proteins, native MIC-1 disappears rapidly from the
bloodstream due to a short plasma half-life, meaning that treatment
with native MIC-1 requires daily administration to maintain
efficacy. The present invention provides compounds comprising MIC-1
fusion proteins with increased plasma half-life.
In what follows, Greek letters may be represented by their symbol
or the corresponding written name, for example: .alpha.=alpha;
.beta.=beta; =epsilon; .gamma.=gamma; .omega.=omega; etc. Also, the
Greek letter of .mu. may be represented by "u", e.g. in .mu.l=ul,
or in .mu.M=uM.
MIC-1 Proteins and Analogues
The term "MIC-1" as used herein means macrophage inhibitory
cytokine-1 (MIC-1), also known as Growth Differentiation Factor 15
(GDF-15), and placental bone morphogenetic protein (PLAB). The
sequence of the full length wild type human MIC-1 protein is
available from the UNIPROT database with accession no. Q99988. The
308 amino acid precursor protein includes a signal peptide (amino
acids 1-29), a propeptide (amino acids 30-196) and a mature protein
(amino acids 197-308). The 112 amino acid mature MIC-1 protein is
included herein as SEQ ID NO:1. Mature MIC-1 contains nine cysteine
residues which give rise to the formation of 4 intrachain
disulphide bonds and one interchain disulphide bond to create a
covalently linked 24.5 kDa homodimer. A naturally occurring
mutation corresponding to His6Asp in the mature protein (SEQ ID
NO:1) has been described.
Thus particular examples of wild type human MIC-1 are the mature
MIC-1 protein of SEQ ID NO:1, SEQ ID NO:1 having the amino acid
modification His6Asp, as well as any of these sequences preceded by
the propeptide and/or signal peptide referred to above.
The term "MIC-1 protein" as used herein refers to the human MIC-1
protein of SEQ ID NO:1, or an analogue thereof. The protein having
the sequence of SEQ ID NO:1 may also be designated "hMIC-1",
"native" MIC-1 or "wild type" MIC-1.
The term "MIC-1 analogue", or "analogue of MIC-1 protein" as used
herein refers to a protein, or a compound, which is a variant of
the mature MIC-1 protein (SEQ ID NO:1). In one aspect, the MIC-1
analogue is a functional variant of the mature MIC-1 protein (SEQ
ID NO:1). In one aspect of the invention, the MIC-1 analogues
display at least 85%, 90% or 95% sequence identity to native MIC-1
(SEQ ID NO:1).
In another aspect of the invention, the MIC-1 analogues comprise
less than 17 amino acid modifications (substitutions, deletions,
additions (including insertions) and any combination thereof)
relative to human native MIC-1 (SEQ ID NO:1).). As an example of a
method for determination of the sequence identity between two
analogues the two peptides His6Asp MIC-1 and native MIC-1 are
aligned. The sequence identity of the His6Asp MIC-1 analogue
relative to native MIC-1 is given by the number of aligned
identical residues minus the number of different residues divided
by the total number of residues in native MIC-1. Accordingly, in
said example the sequence identity in percentage is
(112-1)/112.times.100.
The term "amino acid modification" used throughout this application
is used in the meaning of a modification to an amino acid as
compared to native MIC-1 (SEQ ID NO:1). This modification can be
the result of a deletion of an amino acid, addition of an amino
acid, substitution of one amino acid with another or a substituent
covalently attached to an amino acid of the peptide.
Substitutions.
In one aspect amino acids may be substituted by conservative
substitution. The term "conservative substitution" as used herein
denotes that one or more amino acids are replaced by another,
biologically similar residue. Examples include substitution of
amino acid residues with similar characteristics, e.g. small amino
acids, acidic amino acids, polar amino acids, basic amino acids,
hydrophobic amino acids and aromatic amino acids.
In one aspect amino acids may be substituted by non-conservative
substitution. The term "non-conservative substitution" as used
herein denotes that one or more amino acids are replaced by another
amino acid having different characteristics. Examples include
substitution of a basic amino acid residue with an acidic amino
acid residue, substitution of a polar amino acid residue with an
aromatic amino acid residue, etc. In one aspect, the
non-conservative substitution is substitution of a coded amino acid
to another coded amino acid having different characteristics. In
one aspect, the MIC-1 analogues may comprise substitutions of one
or more unnatural and/or non-amino acids, e.g., amino acid
mimetics, into the sequence of MIC-1.
The asparagine residue in position 3 of human mature MIC-1 (SEQ ID
NO:1) is chemically labile. In one aspect of the invention, the
asparagine in the position corresponding to position 3 of human
mature MIC-1 (SEQ ID NO:1) may be substituted to Ser, Asp, Glu,
Ala, Pro, Thr, Gly, or Gln. In one aspect of the invention, the
asparagine in the position corresponding to position 3 of human
mature MIC-1 (SEQ ID NO:1) has been substituted to Ser. In another
aspect of the invention, the asparagine in the position
corresponding to position 3 of human mature MIC-1 (SEQ ID NO:1) has
been substituted to Glu.
In one aspect of the invention, the arginine in the position
corresponding to position 2 of human mature MIC-1 (SEQ ID NO:1) has
been substituted to alanine.
In one aspect of the invention, the arginine in the position
corresponding to position 2 of human mature MIC-1 (SEQ ID NO:1) has
been substituted to alanine, and the asparagine in the position
corresponding to position 3 of human mature MIC-1 (SEQ ID NO:1) has
been substituted to Glu.
Deletions and Truncations. In one aspect, the MIC-1 analogues of
the invention may have one or more amino acid residues deleted from
the amino acid sequence of human MIC-1, alone or in combination
with one or more insertions or substitutions.
In one aspect, the three N-terminal amino acids of human mature
MIC-1 (Ala1, Arg2, Asn3) may be deleted.
Insertions.
In one aspect, the MIC-1 analogues of the invention may have one or
more amino acid residues inserted into the amino acid sequence of
human MIC-1, alone or in combination with one or more deletions
and/or substitutions.
In one aspect, the MIC-1 analogues of the invention may include
insertions of one or more unnatural amino acids and/or non-amino
acids into the sequence of MIC-1.
MIC-1 analogues may be described by reference to i) the number of
the amino acid residue in the mature MIC-1 protein which
corresponds to the amino acid residue which is changed (i.e., the
corresponding position in native MIC-1), and to ii) the actual
change. In other words, a MIC-1 analogue is a MIC-1 protein in
which a number of amino acid residues have been changed when
compared to native MIC-1 (SEQ ID NO: 1). These changes may
represent, independently, one or more amino acid substitutions,
additions, and/or deletions.
As is apparent from the above examples, amino acid residues may be
identified by their full name, their one-letter code, and/or their
three-letter code. These three ways are fully equivalent.
The term "protein", as e.g. used in the context of MIC-1 proteins,
refers to a compound which comprises a series of amino acids
interconnected by amide (or peptide) bonds.
Amino acids are molecules containing an amine group and a
carboxylic acid group, and, optionally, one or more additional
groups, often referred to as a side chain.
The term "amino acid" includes coded (or proteinogenic or natural)
amino acids (amongst those the 20 standard amino acids), as well as
non-coded (or non-proteinogenic or non-natural) amino acids. Coded
amino acids are those which are naturally incorporated into
proteins. The standard amino acids are those encoded by the genetic
code. Non-coded amino acids are either not found in proteins, or
not produced by standard cellular machinery (e.g., they may have
been subject to post-translational modification). In what follows,
all amino acids of the MIC-1 proteins for which the optical isomer
is not stated is to be understood to mean the L-isomer (unless
otherwise specified).
Human Serum Albumin
Human serum albumin (HSA) belongs to a family of globular proteins
and is composed of 585 amino acids with an approximate molecular
weight of 67 kDa. Albumin comprises three homologous domains that
assemble to form a heart-shaped molecule. Albumin is water-soluble
and soluble in concentrated salt solutions and is commonly found in
blood plasma. Albumin is the most abundant protein of human blood
plasma and its main function is to regulate the osmotic pressure of
blood, transport hormones or fatty acid and buffer pH. The normal
range of human serum albumin in adults is 35 to 50 g/L and human
serum albumin accounts for 80-90% of all plasma protein. As human
serum albumin is a natural carrier for exogenous ligands, it has a
low risk of inducing toxicity and immunogenicity and human serum
albumin extracted from human blood can be used for clinical
purposes. The plasma half-life of human serum albumin is
approximately 20 days. The long half-life of human serum albumin is
caused in part by a pH-dependent recycling mediated by the neonatal
Fc receptor (FcRn). FcRn is present in cells and on the surface of
cells, which interacts with circulating blood, such as vascular
endothelial cells.
Recombinant human serum albumin fusion proteins comprising a
therapeutic protein of interest may be achieved by genetic
manipulation, such that the DNA coding for human serum albumin, or
a fragment thereof, is joined to the DNA encoding for the
therapeutic protein. A suitable expression host is then transformed
or transfected with the fused nucleotide sequences encoded on a
suitable plasmid as to express the fusion protein. Human serum
albumin as fusion partner is thought to increase the plasma
half-life of therapeutic proteins through two biological
mechanisms. The significant size increase inhibits renal clearance
and the inherent ability of human serum albumin to bind the Fc
Neonatal Receptor will allow recycling from the endosome and
prevention of lysomal degradation altogether allowing the molecule
to be present longer in circulation.
Albumin fusion proteins can be produced in expression systems on a
commercial scale and with lower cost than for other methods of
generating therapeutic proteins with long plasma half-lives.
It is known to the person skilled in the art that functional
variants of human serum albumin can be designed, which have the
same plasma half-life prolonging benefits as the wild-type
(truncated and/or amino acid substituted functional variants). For
an example domain III of human serum albumin has been shown to bind
FcN to a high degree and it is possible to make variants comprising
only this domain or combinations with other domains, with long
half-lives or half-lives that are modified (eg. Albufuse Flex
Technology, Novozymes).
The sequence of the wild-type mature human serum albumin is
included herein as SEQ ID NO:2 and the sequence is annotated in the
Uniprot database with the accession no: P02768. The present
invention provides a human serum albumin fusion protein comprising,
or alternatively consisting of, a biologically active MIC-1 protein
or a variant thereof and a biologically active and/or
therapeutically active fragment or variant of human serum albumin.
In one aspect, the invention provides a human serum albumin fusion
protein comprising, or alternatively consisting of, mature native
MIC-1 and the mature native human serum albumin. In one aspect of
the invention, the primary sequence of human serum albumin is
modified. Non-limiting examples includes functional variants of
human serum albumin comprising truncations or amino acid
substitutions or deletions in human serum albumin, which do not
interfere with the half-life extending effect of human serum
albumin. Human serum albumin contains a single thiol group from an
unpaired cysteine residue at position 34 in Domain I. Cys34 in
human serum albumin provides antioxidant activity and constitutes
the largest fraction of free thiol groups in the blood. Cys34-Cys34
disulfide linkage of two human serum albumin molecules has several
disadvantages, which includes side reactions with other residues
during preparation, low stability or structural changes, which
promotes protein aggregation. Substitutions of Cys34 with other
amino acids, such as Ala or Ser has been described previously
(Mccurdy, T et. Al., Journal of Laboratory and Clinical Medicine,
Volume 143, Issue 2, 2004, 115-124). The term "HSA C34A" refers to
a human serum albumin (HSA) variant wherein the cysteine residue at
position 34 of the wild type human serum albumin amino acid
sequence has been replaced with alanine. Other ways of preventing
dimerization and instability through unfavourable interaction of
free Cys at position 34 includes truncation of the N-terminal of
human serum albumin domain I or removal of the Cys residue from the
sequence.
By "functional variant" as used herein is meant a chemical variant
of a certain protein which retains substantially the same function
as the original protein.
Fusion Proteins
"Fusion protein" as used herein is intended to mean a hybrid
protein expressed by a nucleic acid molecule comprising nucleotide
sequences of at least two genes. "Fusion protein" as used herein is
also intended to mean covalent joining of at least two proteins
and/or peptides. In one aspect, the fusion proteins of the
invention comprise human serum albumin as fusion partner fused with
native MIC-1 having an activity of pharmaceutical interest. Fusion
proteins are often used for improving recombinant expression or
stability of therapeutic proteins as well as for improved recovery
and purification of such proteins from cell cultures and the like.
Fusion proteins may comprise artificial sequences, e.g. a linker
sequence.
"Fusion partner" as used herein is intended to mean a protein which
is part of a fusion protein, i.e. one of the at least two proteins
encompassed by the fusion protein.
In one embodiment of the invention the fusion partner comprises
human serum albumin with an approximate molecular weight of 67 kDa
(SEQ ID NO:2) or functional variants thereof, which is operatively
linked to the N-terminal of MIC-1 (SEQ ID NO:1) or functional
variants thereof with a molecular weight of approximately 12 kDa
via an interdomain linker region consisting of amino acid sequences
of different length, charges and/or structural motifs.
"Fusion tag" as used herein is intended to mean a protein sequence
which is part of a fusion protein, i.e. one of the at least two
proteins encompassed by the fusion protein and comprises a sequence
which improves expression, solubilisation or purification of the
fusion protein, e.g. a 6.times. Histidine tag (such as His6) or a
solubilization domain (such as Thiol:disulfide interchange protein
DsbC (DsbC), Maltose Binding Protein (MBP), or Thioredoxin
(Trx)).
In one aspect of the invention, monomers of
NH2-HSA-linker-MIC-1-COOH with a size of approximately 80 kDa,
homodimerizes as the native molecule via interchain disulphide
bridge between the two MIC-1 molecules to form an active HSA-MIC-1
fusion protein with a molecular weight of approximately 160-165 kDa
(depicted as schematic drawing in FIG. 1).
Peptide Linker
The term "peptide linker" as used herein is intended to mean an
amino acid sequence which is typically used to facilitate the
function, folding or expression of fusion proteins.
Different exposure of the MIC-1 protein comprised in a fusion
protein to its putative receptor, plasma half-life or overall
fusion protein stability may be affected by differences in the
linker sequence/structure of the fusion protein, which can cause
changes in biological efficacy, plasma half-life or fusion protein
stability.
The linkers from the present invention were designed with different
predicted biophysical or structural properties comprising
variations in length (variation of the linker length), and
predicted secondary structure such as alpha-helical structure,
rigid structure or flexible, random coil structures or charge. In
the present invention the length of the linker was varied from 7 to
35 amino acids. The linker length may influence the potential
interaction between the human serum albumin and MIC-1 domain by
changing the possibility of steric hindrance provided by the fusion
partner attached to the biological active MIC-1 domain. The steric
hindrance may influence correct folding of the two domains of the
fusion protein monomer, formation of the dimer, the interaction of
the MIC-1 part with a putative receptor, or the linker itself may
interact with either human serum albumin or MIC-1 and that both
composition and length of the linker may in part influence the
nature and extent of such interaction.
Functional Properties
Biological Activity--In Vivo Pharmacology
In one aspect the compounds of the invention are potent in vivo,
which may be determined as is known in the art in any suitable
animal model, as well as in clinical trials.
The non-obese Sprague Dawley rat is one example of a suitable
animal model, and the changes in food intake may be determined in
such rats in vivo, e.g. as described in Example 2.
In one aspect the compounds of the invention inhibits in vivo food
intake in non-obese Sprague Dawley rats.
As an example, in a particular aspect of the invention, the maximum
efficacy which is the greatest significant (p<0.10) reduction in
24 hour food intake recorded over 6-7 days at a dose of 4 nmol/kg
should be more than 20%, preferably more than 30%. In another
particular aspect of the invention, the maximum efficacy which is
the greatest significant (p<0.10) reduction in 24 hour food
intake recorded over 6-7 days at a dose of 4 nmol/kg should be at
least 20%, preferably at least 30%.
As an example, in a particular aspect of the invention, the
accumulated efficacy which is the sum of significant (p<0.10)
reductions in 24 hour food intake compared with vehicle at a dose
of 4 nmol/kg should be more than 50%, more preferably more than
70%, even more preferably more than 80%, or most preferably more
than 100%.
As an example, in a particular aspect of the invention, the
accumulated efficacy which is the sum of significant (p<0.10)
reductions in 24 hour food intake compared with vehicle at a dose
of 4 nmol/kg should be at least 50%, more preferably at least 70%,
even more preferably at least 80%, or most preferably at least
100%.
Diet-Induced Obese (DIO) Sprague Dawley rats is another example of
a suitable animal model, and the changes in food intake may be
determined in such rats in vivo, e.g. as described in Example
3.
In one aspect the compounds of the invention inhibits in vivo food
intake in DIO Sprague Dawley rats.
In one aspect of the invention, the maximum efficacy which is the
greatest significant (p<0.10) reduction in 24 hour food intake
recorded over 6-7 days at a dose of 4 nmol/kg is at least 50%, or
preferably at least 60%.
In one aspect of the invention, the accumulated efficacy which is
the sum of significant (p<0.10) reductions in 24 hour food
intake compared with vehicle at a dose of 4 nmol/kg is at least
300%, more preferably at least 340%, or even more preferably at
least 380%.
Biophysical Properties
In one aspect, the compounds of the invention have good biophysical
properties. These properties include but are not limited to
physical stability and/or solubility. These and other biophysical
properties may be measured using standard methods known in the art.
In a particular embodiment, these properties are improved as
compared to native MIC-1 (SEQ ID NO:1). Increased biophysical
stability of a fusion protein compared to native MIC-1 may be at
least partly be owing to stabilizing effects of the fusion partner
or the length or composition of the intervening amino acid linker
inserted between the human serum albumin and MIC-1 sequence.
Production Processes
Fusion proteins such as those of the present invention may be
produced by means of recombinant protein technology known to
persons skilled in the art. In general, nucleic acid sequences
encoding the proteins of interest or functional variants thereof
are modified to encode the desired fusion protein. This
modification includes the in-frame fusion of the nucleic acid
sequences encoding the two or more proteins to be expressed as a
fusion protein. Such a fusion protein can be with or without a
linker peptide as well as the fusion protein fused to a fusion tag,
e.g. a Histidine tag (such as His6) or a solubilization domain
(such as DsbC, MBP or Trx). This modified sequence is then inserted
into an expression vector, which is in turn transformed or
transfected into the expression host cells.
The nucleic acid construct encoding the fusion protein may suitably
be of genomic, cDNA or synthetic origin. Amino acid sequence
alterations are accomplished by modification of the genetic code by
well-known techniques.
The DNA sequence encoding the fusion protein is usually inserted
into a recombinant vector which may be any vector, which may
conveniently be subjected to recombinant DNA procedures, and the
choice of vector will often depend on the host cell into which it
is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector, which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA
sequence encoding the fusion protein is operably linked to
additional segments required for transcription of the DNA. The
term, "operably linked" indicates that the segments are arranged so
that they function in concert for their intended purposes, e.g.
transcription initiates in a promoter and proceeds through the DNA
sequence coding for the polypeptide until it terminates within a
terminator.
Thus, expression vectors for use in expressing the fusion protein
will comprise a promoter capable of initiating and directing the
transcription of a cloned gene or cDNA. The promoter may be any DNA
sequence, which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell.
Additionally, expression vectors for expression of the fusion
protein will also comprise a terminator sequence, a sequence
recognized by a host cell to terminate transcription. The
terminator sequence is operably linked to the 3' terminus of the
nucleic acid sequence encoding the polypeptide. Any terminator
which is functional in the host cell of choice may be used in the
present invention.
Expression of the fusion protein can be aimed for either
intracellular expression in the cytosol of the host cell or be
directed into the secretory pathway for extracellular expression
into the growth medium.
Intracellular expression is the default pathway and requires an
expression vector with a DNA sequence comprising a promoter
followed by the DNA sequence encoding the fusion protein followed
by a terminator.
To direct the fusion protein into the secretory pathway of the host
cells, a secretory signal sequence (also known as signal peptide or
a pre sequence) is needed as an N-terminal extension of the fusion
protein. A DNA sequence encoding the signal peptide is joined to
the 5' end of the DNA sequence encoding the fusion protein in the
correct reading frame. The signal peptide may be that normally
associated with the protein or may be from a gene encoding another
secreted protein.
The procedures used to ligate the DNA sequences coding for the
fusion protein, the promoter, the terminator and/or secretory
signal sequence, respectively, and to insert them into suitable
vectors containing the information necessary for replication, are
well known to persons skilled in the art (cf., for instance,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, New York, 1989).
The host cell into which the DNA sequence encoding the fusion
protein is introduced may be any cell that is capable of expressing
the fusion protein either intracellularly or extracellularly. The
fusion protein may be produced by culturing a host cell containing
a DNA sequence encoding the fusion protein and capable of
expressing the fusion protein in a suitable nutrient medium under
conditions permitting the expression of the fusion protein.
Non-limiting examples of host cells suitable for expression of
fusion proteins are: Escherichia coli, Saccharomyces cerevisiae, as
well as human embryonic kidney (HEK), Baby Hamster Kidney (BHK) or
Chinese hamster ovary (CHO) cell lines. If posttranslational
modifications are needed, suitable host cells include yeast, fungi,
insects and higher eukaryotic cells such as mammalian cells.
Once the fusion protein has been expressed in a host organism it
may be recovered and purified to the required quality by
conventional techniques. Non-limiting examples of such conventional
recovery and purification techniques are centrifugation,
solubilization, filtration, precipitation, ion-exchange
chromatography, immobilized metal affinity chromatography (IMAC),
Reversed phase--High Performance Liquid Chromatography (RP-HPLC),
gel-filtration and freeze drying.
Examples of recombinant expression and purification of fusion
proteins may be found in e.g. Cordingley et al., 3. Virol. 1989,
63, pp 5037-5045, Birch et al., Protein Expr Purif., 1995, 6, pp
609-618 and in WO2008/043847.
Examples of microbial expression and purification of fusion
proteins may be found in e.g. Chich et al, Anal. Biochem, 1995,
224, pp 245-249 and Xin et al., Protein Expr. Purif. 2002, 24, pp
530-538.
Specific examples of methods of preparing a number of the compounds
of the invention are included in the experimental part.
Mode of Administration
The term "treatment" is meant to include both the prevention and
minimization of the referenced disease, disorder, or condition
(i.e., "treatment" refers to both prophylactic and therapeutic
administration of a compound of the invention or composition
comprising a compound of the invention unless otherwise indicated
or clearly contradicted by context.
The route of administration may be any route which effectively
transports a compound of this invention to the desired or
appropriate place in the body, such as parenterally, for example,
subcutaneously, intramuscularly or intravenously. Alternatively, a
compound of this invention can be administered orally, pulmonary,
rectally, transdermally, buccally, sublingually, or nasally.
The amount of a compound of this invention to be administered, the
determination of how frequently to administer a compound of this
invention, and the election of which compound or compounds of this
invention to administer, optionally together with another
pharmaceutically active agent, is decided in consultation with a
practitioner who is familiar with the treatment of obesity and
related disorders.
Pharmaceutical Compositions
Pharmaceutical compositions comprising a compound of the invention
or a pharmaceutically acceptable salt, amide, or ester thereof, and
a pharmaceutically acceptable excipient may be prepared as is known
in the art.
The term "excipient" broadly refers to any component other than the
active therapeutic ingredient(s). The excipient may be an inert
substance, an inactive substance, and/or a not medicinally active
substance.
The excipient may serve various purposes, e.g. as a carrier,
vehicle, diluent, tablet aid, and/or to improve administration,
and/or absorption of the active substance.
The formulation of pharmaceutically active ingredients with various
excipients is known in the art, see e.g. Remington: The Science and
Practice of Pharmacy (e.g. 19.sup.th edition (1995), and any later
editions).
The term "physical stability" refers to the tendency of the
polypeptide to form biologically inactive and/or insoluble
aggregates as a result of exposure to thermo-mechanical stress,
and/or interaction with destabilising interfaces and surfaces (such
as hydrophobic surfaces). The physical stability of an aqueous
polypeptide formulation may be evaluated by means of visual
inspection, and/or by turbidity measurements after exposure to
mechanical/physical stress (e.g. agitation) at different
temperatures for various time periods. Alternatively, the physical
stability may be evaluated using a spectroscopic agent or probe of
the conformational status of the polypeptide such as e.g.
Thioflavin T or "hydrophobic patch" probes.
The term "chemical stability" refers to chemical (in particular
covalent) changes in the polypeptide structure leading to formation
of chemical degradation products potentially having a reduced
biological potency, and/or increased immunogenic effect as compared
to the intact polypeptide. The chemical stability can be evaluated
by measuring the amount of chemical degradation products at various
time-points after exposure to different environmental conditions,
e.g. by SEC-HPLC, and/or RP-HPLC.
Combination Treatment
The treatment with a compound according to the present invention
may also be combined with one or more pharmacologically active
substances, e.g., selected from antiobesity agents, appetite
regulating agents, and agents for the treatment and/or prevention
of complications and disorders resulting from or associated with
obesity.
Pharmaceutical Indications
In one aspect, the present invention relates to a compound of the
invention, for use as a medicament.
In particular embodiments, the compound of the invention may be
used for the following medical treatments:
(i) Prevention and/or treatment of eating disorders, such as
obesity, e.g. by decreasing food intake, reducing body weight,
suppressing appetite and inducing satiety.
(ii) Prevention and/or treatment of hyperglycemia and/or impaired
glucose tolerance.
In some embodiments the invention relates to a method for weight
management. In some embodiments the invention relates to a method
for reduction of appetite. In some embodiments the invention
relates to a method for reduction of food intake.
Generally, all subjects suffering from obesity are also considered
to be suffering from overweight. In some embodiments the invention
relates to a method for treatment or prevention of obesity. In some
embodiments the invention relates to use of the MIC-1 fusion
proteins of the invention for treatment or prevention of obesity.
In some embodiments the subject suffering from obesity is human,
such as an adult human or a paediatric human (including infants,
children, and adolescents). Body mass index (BMI) is a measure of
body fat based on height and weight. The formula for calculation is
BMI=weight in kilograms/height in meters2. A human subject
suffering from obesity may have a BMI of .gtoreq.30; this subject
may also be referred to as obese. In some embodiments the human
subject suffering from obesity may have a BMI of .gtoreq.35 or a
BMI in the range of .gtoreq.30 to <40. In some embodiments the
obesity is severe obesity or morbid obesity, wherein the human
subject may have a BMI of .gtoreq.40.
In some embodiments the invention relates to a method for treatment
or prevention of overweight, optionally in the presence of at least
one weight-related comorbidity. In some embodiments the invention
relates to use of the MIC-1 fusion proteins of the invention for
treatment or prevention of overweight, optionally in the presence
of at least one weight-related comorbidity.
In some embodiments the subject suffering from overweight is human,
such as an adult human or a paediatric human (including infants,
children, and adolescents). In some embodiments a human subject
suffering from overweight may have a BMI of .gtoreq.25, such as a
BMI of .gtoreq.27. In some embodiments a human subject suffering
from overweight has a BMI in the range of 25 to <30 or in the
range of 27 to <30. In some embodiments the weight-related
comorbidity is selected from the group consisting of hypertension,
diabetes (such as type 2 diabetes), dyslipidaemia, high
cholesterol, and obstructive sleep apnoea.
In some embodiments the invention relates to a method for reduction
of body weight. In some embodiments the invention relates to use of
the MIC-1 fusion proteins of the invention for reduction of body
weight. A human to be subjected to reduction of body weight
according to the present invention may have a BMI of .gtoreq.25,
such as a BMI of 27 or a BMI of .gtoreq.30. In some embodiments the
human to be subjected to reduction of body weight according to the
present invention may have a BMI of .gtoreq.35 or a BMI of
.gtoreq.40. The term "reduction of body weight" may include
treatment or prevention of obesity and/or overweight.
PARTICULAR EMBODIMENTS
The invention is further described by the following non-limiting
embodiments of the invention:
1. A compound comprising a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin or a functional variant
thereof; B is a peptide linker comprising the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is at least 2; and C is a MIC-1 protein or an analogue
thereof, and wherein the C-terminus of human serum albumin or a
functional variant thereof is fused to the N-terminus of the
peptide linker, and the C-terminus of the peptide linker is fused
to the N-terminus of the MIC-1 protein or analogue thereof. 2. A
compound consisting of a fusion protein of formula (I): A-B-C (I),
wherein A is human serum albumin or a functional variant thereof; B
is a peptide linker comprising the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is at least 2; and C is a MIC-1 protein or an analogue
thereof, and wherein the C-terminus of human serum albumin or a
functional variant thereof is fused to the N-terminus of the
peptide linker, and the C-terminus of the peptide linker is fused
to the N-terminus of the MIC-1 protein or analogue thereof. 3. A
compound comprising a fusion protein of formula (I): A-B-C (I),
wherein A is human serum albumin or a functional variant thereof; B
is a peptide linker, wherein the peptide linker is 10 to 50 amino
acids in length and comprises the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is at least 2; and C is a MIC-1 protein or an analogue
thereof, and wherein the C-terminus of human serum albumin or a
functional variant thereof is fused to the N-terminus of the
peptide linker, and the C-terminus of the peptide linker is fused
to the N-terminus of the MIC-1 protein or analogue thereof. 4. A
compound consisting of a fusion protein of formula (I): A-B-C (I),
wherein A is human serum albumin or a functional variant thereof; B
is a peptide linker, wherein the peptide linker is 10 to 50 amino
acids in length and comprises the amino acid sequence
[X-Y.sub.m].sub.n, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is at least 2; and C is a MIC-1 protein or an analogue
thereof, and wherein the C-terminus of human serum albumin or a
functional variant thereof is fused to the N-terminus of the
peptide linker, and the C-terminus of the peptide linker is fused
to the N-terminus of the MIC-1 protein or analogue thereof. 5. A
compound according to any one of the preceding embodiments, wherein
the compound is a homodimer of two fusion proteins of formula (I):
A-B-C (I) formed by an interchain disulphide bridge between the two
MIC-1 proteins or analogues thereof. 6. A compound according to any
one of the preceding embodiments, wherein the peptide linker is 10
to 35 amino acids in length. 7. A compound according to any one of
the preceding embodiments, wherein the peptide linker is 15 to 25
amino acids in length. 8. A compound according to any one of the
preceding embodiments, wherein the peptide linker is 20 to 25 amino
acids in length. 8a. A compound according to any one of the
preceding embodiments, wherein the peptide linker is 20 to 30 amino
acids in length. 9. A compound according to any one of the
preceding embodiments, wherein the peptide linker consists of a
maximum of 35 amino acids. 10. A compound according to any one of
the preceding embodiments, wherein the peptide linker consists of a
maximum of 30 amino acids. 11. A compound according to any one of
the preceding embodiments, wherein the peptide linker consists of a
maximum of 25 amino acids. 12. A compound according to any one of
the preceding embodiments, wherein the peptide linker comprises the
amino acid sequence [X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y
is Ala; m is from 2 to 4; and n is at least 2. 13. A compound
according to any one of the preceding embodiments, wherein the
peptide linker has the amino acid sequence [X-Y.sub.m].sub.nX,
wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at
least 2. 14. A compound according to any one of the preceding
embodiments, wherein the peptide linker has the amino acid sequence
[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is from 2
to 4; and n is at least 5. 15. A compound according to any one of
the preceding embodiments, wherein the peptide linker has the amino
acid sequence [X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is
Ala; m is from 2 to 3; and n is at least 5. 16. A compound
according to any one of the preceding embodiments, wherein the
peptide linker comprises the amino acid sequence
GGSS[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is
from 2 to 4; and n is at least 2 (wherein GGSS is SEQ ID NO: 40).
17. A compound according to any one of the preceding embodiments,
wherein the peptide linker has the amino acid sequence
GGSS[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is
from 2 to 4; and n is at least 2 (wherein GGSS is SEQ ID NO: 40).
18. A compound according to any one of the preceding embodiments,
wherein the peptide linker has the amino acid sequence
GGSS[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is
from 2 to 4; and n is at least 5 (wherein GGSS is SEQ ID NO: 40).
19. A compound according to any one of the preceding embodiments,
wherein the peptide linker has the amino acid sequence
GGSS[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is
from 2 to 3; and n is at least 5 (wherein GGSS is SEQ ID NO: 40).
20. A compound according to any one of the preceding embodiments,
wherein the peptide linker has the amino acid sequence
GGSS[X-Y.sub.m].sub.nX, wherein X is Asp or Glu; Y is Ala; m is 2;
and n is 5 or 6 (wherein GGSS is SEQ ID NO: 40). 21. A compound
according to any one of the preceding embodiments, wherein the
peptide linker has the amino acid sequence GGSS[X-Y.sub.m].sub.nX,
wherein X is Asp or Glu; Y is Ala; m is 2; and n is 6 (wherein GGSS
is SEQ ID NO: 40). 22. A compound according to any one of the
preceding embodiments, wherein X is Asp. 23. A compound according
to any one of the preceding embodiments, wherein X is Glu. 24. A
compound according to any one of the preceding embodiments, wherein
m is 2 and n is 2, 4 or 6. 25. A compound according to any one of
the preceding embodiments, wherein n is 2, 4 or 6. 26. A compound
according to any one of the preceding embodiments, wherein n is 6.
27. A compound according to any one of the preceding embodiments,
wherein m is 2. 27a. A compound according to any one of the
preceding embodiments, wherein m is 3. 28. A compound according to
any one of the preceding embodiments, wherein the peptide linker
comprises (Glu-Ala-Ala).sub.6 (SEQ ID NO: 39). 29. A compound
according to any one of the preceding embodiments, wherein the
peptide linker is (Glu-Ala-Ala).sub.6 (SEQ ID NO: 39). 30. A
compound according to any one of the preceding embodiments, wherein
the peptide linker comprises (Glu-Ala-Ala).sub.6-Glu (SEQ ID NO:
11). 31. A compound according to any one of the preceding
embodiments, wherein the peptide linker is (Glu-Ala-Ala).sub.6-Glu
(SEQ ID NO: 11). 32. A compound according to any one of the
preceding embodiments, wherein the peptide linker comprises
Gly-Gly-Ser-Ser-(Glu-Ala-Ala).sub.6-Glu (SEQ ID NO: 9). 33. A
compound according to any one of the preceding embodiments, wherein
the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala).sub.6-Glu (SEQ
ID NO: 9). 34. A compound according to any one of the preceding
embodiments, wherein the peptide linker comprises
(Glu-Ala-Ala).sub.10-Glu (SEQ ID NO: 12). 35. A compound according
to any one of the preceding embodiments, wherein the peptide linker
is (Glu-Ala-Ala).sub.10-Glu (SEQ ID NO: 12). 36. A compound
according to any one of the preceding embodiments, wherein the
peptide linker comprises Gly-Gly-Ser-Ser-(Glu-Ala-Ala).sub.10-Glu
(SEQ ID NO: 13). 37. A compound according to any one of the
preceding embodiments, wherein the peptide linker is
Gly-Gly-Ser-Ser-(Glu-Ala-Ala).sub.10-Glu (SEQ ID NO: 13). 38. A
compound according to any one of the preceding embodiments, wherein
the peptide linker comprises (Glu-Ala-Ala-Ala).sub.5-Glu (SEQ ID
NO: 33). 39. A compound according to any one of the preceding
embodiments, wherein the peptide linker is
(Glu-Ala-Ala-Ala).sub.5-Glu (SEQ ID NO: 33). 40. A compound
according to any one of the preceding embodiments, wherein the
peptide linker comprises
Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala).sub.5-Glu (SEQ ID NO: 35). 41. A
compound according to any one of the preceding embodiments, wherein
the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala).sub.5-Glu
(SEQ ID NO: 35). 42. A compound according to any one of the
preceding embodiments, wherein the peptide linker comprises
(Glu-Ala-Ala-Ala).sub.6-Glu (SEQ ID NO: 36). 43. A compound
according to any one of the preceding embodiments, wherein the
peptide linker is (Glu-Ala-Ala-Ala).sub.6-Glu (SEQ ID NO: 36). 44.
A compound according to any one of the preceding embodiments,
wherein the peptide linker comprises
Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala).sub.6-Glu (SEQ ID NO: 38). 45. A
compound according to any one of the preceding embodiments, wherein
the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala).sub.6-Glu
(SEQ ID NO: 38). 46. A compound according to any one of the
preceding embodiments, wherein the peptide linker comprises
(Asp-Ala-Ala).sub.6-Asp (SEQ ID NO: 10). 47. A compound according
to any one of the preceding embodiments, wherein the peptide linker
is (Asp-Ala-Ala).sub.6-Asp (SEQ ID NO: 10). 48. A compound
according to any one of the preceding embodiments, wherein the
peptide linker comprises (Asp-Ala-Ala-Ala).sub.5-Asp (SEQ ID NO:
34). 49. A compound according to any one of the preceding
embodiments, wherein the peptide linker is
(Asp-Ala-Ala-Ala).sub.5-Asp (SEQ ID NO: 34). 50. A compound
according to any one of the preceding embodiments, wherein the
peptide linker comprises (Asp-Ala-Ala-Ala).sub.6-Asp (SEQ ID NO:
37). 51. A compound according to any one of the preceding
embodiments, wherein C is an analogue of MIC-1 displaying at least
85% sequence identity to native MIC-1 (SEQ ID NO:1). 52. A compound
according to any one of the preceding embodiments, wherein C is an
analogue of MIC-1 displaying at least 90% sequence identity to
native MIC-1 (SEQ ID NO:1). 53. A compound according to any one of
the preceding embodiments, wherein C is an analogue of MIC-1
displaying at least 95% sequence identity to native MIC-1 (SEQ ID
NO:1). 54. A compound according to any one of the preceding
embodiments, wherein C is an analogue of MIC-1 having a maximum of
17 amino acid modifications compared to native MIC-1 (SEQ ID NO:1).
55. A compound according to any one of the preceding embodiments,
wherein C is an analogue of MIC-1 having a maximum of 11 amino acid
modifications compared to native MIC-1 (SEQ ID NO:1). 56. A
compound according to any one of the preceding embodiments, wherein
C is an analogue of MIC-1 having a maximum of 5 amino acid
modifications compared to native MIC-1 (SEQ ID NO:1). 57. A
compound according to any one of the preceding embodiments, wherein
C is mature human MIC-1 (SEQ ID NO:1). 58. A compound according to
any one of the preceding embodiments, wherein C is N3S hMIC-1 of
SEQ ID NO:14. 59. A compound according to any one of the preceding
embodiments, wherein C is R2A, N3E hMIC-1 of SEQ ID NO:15. 60. A
compound according to any one of the preceding embodiments, wherein
C is N3E hMIC-1 of SEQ ID NO:16. 61. A compound according to any
one of the preceding embodiments, wherein C is N3A hMIC-1 of SEQ ID
NO:17. 62. A compound according to any one of the preceding
embodiments, wherein C is N3P hMIC-1 of SEQ ID NO:18. 63. A
compound according to any one of the preceding embodiments, wherein
C is N3T hMIC-1 of SEQ ID NO:19. 64. A compound according to any
one of the preceding embodiments, wherein C is N3G hMIC-1 of SEQ ID
NO:20. 65. A compound according to any one of the preceding
embodiments, wherein C is N3Q hMIC-1 of SEQ ID NO:21. 66. A
compound according to any one of the preceding embodiments, wherein
C is N3D hMIC-1 of SEQ ID NO:22. 67. A compound according to any
one of the preceding embodiments, wherein C is SEQ ID NO:14. 68. A
compound according to any one of the preceding embodiments, wherein
C is SEQ ID NO:15. 69. A compound according to any one of the
preceding embodiments, wherein C is SEQ ID NO:16. 70. A compound
according to any one of the preceding embodiments, wherein C is SEQ
ID NO:17. 71. A compound according to any one of the preceding
embodiments, wherein C is SEQ ID NO:18. 72. A compound according to
any one of the preceding embodiments, wherein C is SEQ ID NO:19.
73. A compound according to any one of the preceding embodiments,
wherein C is SEQ ID NO:20. 74. A compound according to any one of
the preceding embodiments, wherein C is SEQ ID NO:21. 75. A
compound according to any one of the preceding embodiments, wherein
C is SEQ ID NO:22. 76. A compound according to any one of the
preceding embodiments, wherein A is wild type human serum albumin
of SEQ ID NO:2. 77. A compound according to any one of the
preceding embodiments, wherein A is an analogue of human serum
albumin displaying at least 85% sequence identity to wild type
human serum albumin of SEQ ID NO:2. 78. A compound according to any
one of the preceding embodiments, wherein A is an analogue of human
serum albumin displaying at least 90% sequence identity to wild
type human serum albumin of SEQ ID NO:2. 79. A compound according
to any one of the preceding embodiments, wherein A is an analogue
of human serum albumin displaying at least 95% sequence identity to
wild type human serum albumin of SEQ ID NO:2. 80. A compound
according to any one of the preceding embodiments, wherein A is
C34A human serum albumin of SEQ ID NO:23. 81. A compound according
to any one of the preceding embodiments, wherein A is SEQ ID NO:23.
82. A compound according to any one of the preceding embodiments,
further comprising a fusion partner. 83. A compound according to
any one of the preceding embodiments, further comprising an
N-terminal fusion partner. 84. A compound according to any one of
the preceding embodiments, wherein said compound is a MIC-1
agonist. 85. A compound according to any one of the preceding
embodiments, wherein said compound is capable of decreasing food
intake. 86. A compound according to any one of the preceding
embodiments, wherein said compound has the effect in vivo of
decreasing food intake determined in a single-dose study in
non-obese Sprague Dawley rats. 87. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), selected from the following: A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:10, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:11, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:33, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:34, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:9, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:35, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:36, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:37, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:38, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:12, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:13, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
native human MIC-1 of SEQ ID NO:1; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:14; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:15; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:16; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:17; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:18; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:19; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:20; A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:21; and A is human serum albumin
protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,
and C is the MIC-1 variant of SEQ ID NO:22. 88. A compound
according to embodiment 1, consisting of a fusion protein of
formula (I): A-B-C (I), selected from the following: A is human
serum albumin protein of SEQ ID NO:2, B is the
peptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ
ID NO:1; A is human serum albumin protein of SEQ ID NO:23, B is the
peptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ
ID NO:1; A is human serum albumin protein of SEQ ID NO:23, B is the
peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID
NO:14; and A is human serum albumin protein of SEQ ID NO:23, B is
the peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of
SEQ ID NO:15. 89. A compound according to embodiment 1, wherein A
is human serum albumin protein of SEQ ID NO:2, B is the peptide
linker of SEQ ID NO:10, and C is native human MIC-1 of SEQ ID NO:1.
90. A compound according to embodiment 1, wherein A is human serum
albumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID
NO:11, and C is native human MIC-1 of SEQ ID NO:1. 91. A compound
according to embodiment 1, wherein A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:33, and C is
native human MIC-1 of SEQ ID NO:1. 92. A compound according to
embodiment 1, wherein A is human serum albumin protein of SEQ ID
NO:2, B is the peptide linker of SEQ ID NO:34, and C is native
human MIC-1 of SEQ ID NO:1. 93. A compound according to embodiment
1, wherein A is human serum albumin protein of SEQ ID NO:2, B is
the peptide linker of SEQ ID NO:9, and C is native human MIC-1 of
SEQ ID NO:1. 94. A compound according to embodiment 1, wherein A is
human serum albumin protein of SEQ ID NO:2, B is the peptide linker
of SEQ ID NO:35, and C is native human MIC-1 of SEQ ID NO:1. 95. A
compound according to embodiment 1, wherein A is human serum
albumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID
NO:36, and C is native human MIC-1 of SEQ ID NO:1. 96. A compound
according to embodiment 1, wherein A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:37, and C is
native human MIC-1 of SEQ ID NO:1. 97. A compound according to
embodiment 1, wherein A is human serum albumin protein of SEQ ID
NO:2, B is the peptide linker of SEQ ID NO:38, and C is native
human MIC-1 of SEQ ID NO:1. 98. A compound according to embodiment
1, wherein A is human serum albumin protein of SEQ ID NO:2, B is
the peptide linker of SEQ ID NO:12, and C is native human MIC-1 of
SEQ ID NO:1. 99. A compound according to embodiment 1, wherein A is
human serum albumin protein of SEQ ID NO:2, B is the peptide linker
of SEQ ID NO:13, and C is native human MIC-1 of SEQ ID NO:1. 100. A
compound according to embodiment 1, wherein A is human serum
albumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID
NO:9, and C is native human MIC-1 of SEQ ID NO:1. 101. A compound
according to embodiment 1, wherein A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:14. 102. A compound according to
embodiment 1, wherein A is human serum albumin protein of SEQ ID
NO:23, B is the peptide linker of SEQ ID NO:9, and C is the MIC-1
variant of SEQ ID NO:15. 103. A compound according to embodiment 1,
wherein A is human serum albumin protein of SEQ ID NO:23, B is the
peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID
NO:16. 104. A compound according to embodiment 1, wherein A is
human serum albumin protein of SEQ ID NO:23, B is the peptide
linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:17.
105. A compound according to embodiment 1, wherein A is human serum
albumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID
NO:9, and C is the MIC-1 variant of SEQ ID NO:18. 106. A compound
according to embodiment 1, wherein A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:19. 107. A compound according to
embodiment 1, wherein A is human serum albumin protein of SEQ ID
NO:23, B is the peptide linker of SEQ ID NO:9, and C is the MIC-1
variant of SEQ ID NO:20. 108. A compound according to embodiment 1,
wherein A is human serum albumin protein of SEQ ID NO:23, B is the
peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID
NO:21. 109. A compound according to embodiment 1, wherein A is
human serum albumin protein of SEQ ID NO:23, B is the peptide
linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:22.
110. A compound according to embodiment 1, consisting of a fusion
protein of formula (I): A-B-C (I), wherein A is human serum albumin
protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:10,
and C is native human MIC-1 of SEQ ID NO:1. 111. A compound
according to embodiment 1, consisting of a fusion protein of
formula (I): A-B-C (I), wherein A is human serum albumin protein of
SEQ ID NO:2, B is the peptide linker of SEQ ID NO:11, and C is
native human MIC-1 of SEQ ID NO:1. 112. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:2, B is
the peptide linker of SEQ ID NO:33, and C is native human MIC-1 of
SEQ ID NO:1. 113. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:2, B is the peptide linker of
SEQ ID NO:34, and C is native human MIC-1 of SEQ ID NO:1. 114. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:9, and C is
native human MIC-1 of SEQ ID NO:1. 115. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:2, B is
the peptide linker of SEQ ID NO:35, and C is native human MIC-1 of
SEQ ID NO:1. 116. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:2, B is the peptide linker of
SEQ ID NO:36, and C is native human MIC-1 of SEQ ID NO:1. 117. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:37, and C is
native human MIC-1 of SEQ ID NO:1. 118. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:2, B is
the peptide linker of SEQ ID NO:38, and C is native human MIC-1 of
SEQ ID NO:1. 119. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:2, B is the peptide linker of
SEQ ID NO:12, and C is native human MIC-1 of SEQ ID NO:1. 120. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:13, and C is
native human MIC-1 of SEQ ID NO:1. 121. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:23, B is
the peptide linker of SEQ ID NO:9, and C is native human MIC-1 of
SEQ ID NO:1. 122. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:23, B is the peptide linker of
SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:14. 123. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:15. 124. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:23, B is
the peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of
SEQ ID NO:16. 125. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:23, B is the peptide linker of
SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:17. 126. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:18. 127. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:23, B is
the peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of
SEQ ID NO:19. 128. A compound according to embodiment 1, consisting
of a fusion protein of formula (I): A-B-C (I), wherein A is human
serum albumin protein of SEQ ID NO:23, B is the peptide linker of
SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:20. 129. A
compound according to embodiment 1, consisting of a fusion protein
of formula (I): A-B-C (I), wherein A is human serum albumin protein
of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9, and C is
the MIC-1 variant of SEQ ID NO:21. 130. A compound according to
embodiment 1, consisting of a fusion protein of formula (I): A-B-C
(I), wherein A is human serum albumin protein of SEQ ID NO:23, B is
the peptide linker of SEQ ID NO:9, and C is the MIC-1 variant of
SEQ ID NO:22. 131. A compound according to embodiment 1, selected
from the following: compound 23, compound 24, and compound 26. 132.
A compound according to embodiment 1, wherein the compound is
compound 23. 133. A compound according to embodiment 1, wherein the
compound is compound 24. 134. A compound according to embodiment 1,
wherein the compound is compound 25. 135. A compound according to
embodiment 1, wherein the compound is compound 26. 136. A compound
according to embodiment 1, consisting of a His-tagged fusion
protein of formula (I), wherein the His-tag is the His-tag of SEQ
ID NO:3, A is human serum albumin protein of SEQ ID NO:2, B is the
peptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ
ID NO:1. 137. A compound according to embodiment 1, consisting of a
His-tagged fusion protein of formula (I), wherein the His-tag is
the His-tag of SEQ ID NO:3, A is human serum albumin protein of SEQ
ID NO:2, B is the peptide linker of SEQ ID NO:10, and C is native
human MIC-1 of SEQ ID NO:1. 138. A compound according to embodiment
1, consisting of a His-tagged fusion protein of formula (I),
wherein the His-tag is the His-tag of SEQ ID NO:3, wherein A is
human serum albumin protein of SEQ ID NO:2, B is the peptide linker
of SEQ ID NO:11, and C is native human MIC-1 of SEQ ID NO:1. 139. A
compound according to embodiment 1, consisting of a His-tagged
fusion protein of formula (I), wherein the His-tag is the His-tag
of SEQ ID NO:3, wherein A is human serum albumin protein of SEQ ID
NO:2, B is the peptide linker of SEQ ID NO:12, and C is native
human MIC-1 of SEQ ID NO:1. 140. A compound according to embodiment
1, consisting of a His-tagged fusion protein of formula (I),
wherein the His-tag is the His-tag of SEQ ID NO:3, wherein A is
human serum albumin protein of SEQ ID NO:2, B is the peptide linker
of SEQ ID NO:13, and C is native human MIC-1 of SEQ ID NO:1. 141. A
pharmaceutical composition comprising a compound according to any
one of embodiments 1-140 or a pharmaceutically acceptable salt,
amide or ester thereof, and one or more pharmaceutically acceptable
excipients. 142. A compound according to any one of embodiments
1-140 for use as a medicament. 143. A compound according to any one
of embodiments 1-140 for use in the prevention and/or treatment of
eating disorders, such as obesity, e.g. by decreasing food intake,
reducing body weight, suppressing appetite and inducing satiety.
144. A compound according to any one of embodiments 1-140 for use
in the prevention and/or treatment of obesity. 145. The use of a
compound according to any one of embodiments 1-140 in the
manufacture of a medicament for the treatment of eating disorders,
such as obesity, e.g. by decreasing food intake, reducing body
weight, suppressing appetite and inducing satiety. 146. The use of
a compound according to any one of embodiments 1-140 in the
manufacture of a medicament for the treatment of obesity. 147. A
method of treating or preventing eating disorders, such as obesity,
e.g. by decreasing food intake, reducing body weight, suppressing
appetite and inducing satiety by administering a pharmaceutically
active amount of a compound according to any one of embodiments
1-140. 148. A method of treating or preventing obesity by
administering a pharmaceutically active amount of a compound
according to any one of embodiments 1-140. 149. A polynucleotide
molecule encoding a compound according to any one of embodiments
1-140.
EXAMPLES
This experimental part starts with a list of abbreviations, and is
followed by a section including general methods of preparation,
purification and characterisation of the compounds of the
invention. Then follows an example relating to the activity and
properties of these fusion proteins (section headed pharmacological
methods). The examples serve to illustrate the invention.
LIST OF ABBREVIATIONS
"Main peak" refers to the peak in a purification chromatogram which
has the highest UV intensity in milliabsorbance units and which
contains the fusion protein.
HPLC is High performance liquid chromatography.
SDS-PAGE is Sodium dodecyl sulfate Polyacrylamide gel
electrophoresis.
IMAC is immobilized metal affinity chromatography.
SEC is size exclusion chromatography.
MS is mass spectrometry.
Materials and Methods
General Methods of Preparation
General Expression Method 1: Small Scale Screening and Expression
of Fusion Constructs
Expression levels for each construct were determined by transient
transfection of the plasmids into Human Embryonic Kidney (HEK)
cells (Expi293F.TM., Life Technologies.TM. #A14527) in 2 ml
suspension cultures grown in Expi293.TM. Expression Medium (Life
Technologies.TM. #A1435101). The expi293 cells were grown in
disposable 24-well multiwell blocks (Axygen, #P-DW-10 ml-24-C-S) at
37.degree. C., 8% CO.sub.2 and 80% humidity. The shaking speed was
200 rpm in an Infors Multitron Cell incubator with a 50 mm orbital
throw. For each transfection, 2 .mu.g DNA in 100 ul of transfection
medium (Opti-MEM.RTM. I (1.times.)+GlutaMAX.TM.-I Reduced Serum
Medium, Life Technologies.TM. #51985-026) and 5.4 .mu.l
ExpiFectamine.TM. 293 reagent (ExpiFectamine.TM. 293 Transfection
Kit, Life Technologies.TM. #A14525) in transfection medium were
used, according to the manufacturer's instructions. 18 hours after
transfection, the cultures were fed with 10 .mu.l enhancer 1 and
100 .mu.l enhancer 2 (ExpiFectamine.TM. 293 Transfection Kit, Life
Technologies.TM. #A14525). Approximately 90 hours after
transfection, the cell cultures were harvested by centrifugation at
4000 g for 10 minutes, and the clarified culture medium used for
further analysis of protein expression.
The relative expression levels of the constructs were determined by
loading clarified cell supernatants directly on SDS-PAGE (Sodium
dodecyl sulfate Polyacrylamide gel electrophoresis) gels
(Novex.RTM. NuPAGE.RTM. 4-12% Bis-Tris midi protein gels, 26 wells,
Life Technologies.TM. #WG1403BOX) without sample reduction, and the
resulting protein bands visualized by Coomassie staining
(InstantBlue.TM., Expedeon #ISBL1L). The production feasibility of
each fusion protein was assessed by a small scale purification
screen using an immobilized metal affinity chromatography (IMAC)
step. The purified protein solutions were visualized by SDS-PAGE
and Coomassie staining as described above and the results were used
to determine the cell culture volume needed of each construct to
provide enough protein for in vivo assessment of efficacy.
General Expression Method 2: Scale-Up Expression of his-Tagged
HSA-MIC-1 Fusion Proteins
The plasmids encoding MIC-1 fusion proteins were transformed to
OneShot.RTM. Top10F'' chemically competent E. coli cells (Life
Technologies.TM. #C303003), colonies were grown on Amp/Carb
selective agar plates and transformants used to inoculate liquid
Terrific Broth (TB) cultures. After overnight growth, the pelleted
E. coli cells were used for large scale plasmid preparations
(EndoFree.RTM. Plasmid Mega Kit, Qiagen.RTM. #12381).
Transient expression was performed by adding plasmid DNA (1
mg/liter cell culture) in OptiMEM.RTM. transfection medium (50
ml/liter cell culture) to ExpiFectamine.TM. 293 reagent (2.7
ml/liter cell culture) in OptiMEM.RTM. transfection medium (50
ml/liter cell culture), incubating for 20 minutes and then adding
the transfection mix to the cell culture (expi293F cells at
3.times.106 cells/ml). 18 hours after transfection, the cultures
were fed with enhancers 1 (5 ml/liter cell culture) and enhancer 2
(50 ml/liter cell culture). The expi293 cells were grown in 1 liter
disposable shaker flasks (Corning #CLS431147) at 37.degree. C., 8%
CO.sub.2 and 80% humidity. The shaking speed was 110 rpm in an
Infors Multitron Cell incubator with a 50 mm orbital throw.
Approximately 90 hours after transfection the cultures were
harvested by centrifugation at 4000 g for 10 minutes. The clarified
medium was sterile filtered through a 0.22 uM filter before
purification.
Purification
Following centrifugation and filtration through a 0.22 .mu.m PES
Bottle-top filter (Techno Plastic Products AG, Switzerland) the
clarified supernatant was conditioned for IMAC purification by
addition of 200 mL His-binding buffer (300 mM Sodium Phosphate
(NaP), 1.8 M NaCl, 60 mM imidazole, pH 7.5) per liter
supernatant.
Using an AKTAxpress chromatography system, the conditioned
supernatant was applied at low flowrate to a 5 ml HisTrap Excel
column (GE-Healthcare, Sweden) equilibrated in Buffer A (50 mM NaP,
300 mM NaCl, 10 mM Imidazole, pH 7.5) after which low affinity
binding impurities were eluted with Wash Buffer (50 mM NaP, 300 mM
NaCl, 30 mM Imidazole, pH 7.5). Bound fusion protein was step
eluted with 100% Buffer B (50 mM NaP, 300 mM NaCl, 500 mM
Imidazole, pH 7.5) and the main peak was collected using the peak
detection option of the Unicorn.TM. software and automatically
purified further using preparative size exclusion chromatography
(SEC) in 1.times.PBS pH 7.4 (Ampliqon) on a HiLoad Superdex 200
16/600 PG column (GE-Healthcare, Sweden). 1.5 ml fractions were
collected and analyzed by reducing and non-reducing SDS-PAGE using
precast 4-12% NuPAGE.RTM. gels (Life Technologies.TM.). In short
the samples were mixed with 4.times.LDS (lithium dodecyl sulphate)
sample buffer supplemented with 10.times. reducing agent when
reduction was required. The mixture was heated 5 minutes at
95.degree. C. before loading the SDS-PAGE gels. Novex SeeBlue.RTM.
plus2 pre-stained Protein standard (Life Technologies.TM.) were run
alongside the fractions on SDS-PAGE for size estimation. Protein
was visualized using InstantBlue.TM. stain (Expedeon,
Cambridgeshire, UK) according to the manufacturer instructions.
General Expression Method 3: Recombinant Expression of
HSA-Linker-MIC-1 Fusion Protein
Generation of Vectors for Recombinant Expression of
HSA-Linker-MIC-1 Fusion Proteins:
A series of CMV promoter-based expression vectors (pTT vectors)
were generated for transient expression of HSA-linker-MIC-1 fusion
proteins in EXPI293F cells (Life Technologies). The pTT vectors
were generated for transient protein expression in the HEK293-6E
EBNA-based expression system developed by Yves Durocher (Durocher
et al. Nucleic Acid Research, 2002) and can be used for transient
expression in the Expi293 expression system.
Initially, the gene constructs of each Albumin-linker-MIC-1 fusion
protein variant were ordered in pTT vectors at Genscript with the
human CD33 signal peptide sequence. The plasmids were subsequently
transformed into E. coli for selection and the sequences of the
constructs were verified by DNA sequencing.
Recombinant Expression of Fusion Proteins:
The HSA-linker-MIC-1 fusion proteins were expressed transiently in
EXPI293F cells (Life 30 Technologies) by transfection of the
pTT-based expression vectors according to manufacturer's
instructions. The following procedure describes the generic
EXPI293F expression protocol.
Cell Maintenance:
EXPI293F cells were grown in suspension in Expi293.TM. expression
medium (Life Technologies). Cells were cultured in Erlenmeyer
shaker flasks in an orbital shaker incubator at 36.5.degree. C., 8%
CO2 and 85-125 rpm and maintained at cell densities between
0.4-4.times.10E6 cells/mL.
DNA Transfection:
Typically, 30-1000 mL culture volumes were transfected. Separate
dilutions of DNA and transfection reagent were initially prepared.
Following components were mixed per 1-mL cell culture: 1. A total
of 1 .mu.g vector DNA was diluted in 50 .mu.L Opti-MEM media
(Gibco) and incubated at room temperature (23-25.degree. C.) for 5
min. 2. A total of 2.7 .mu.L Expifectamin.TM. 293 (Life
Technologies) was diluted in 50 .mu.L Opti-MEM media (Gibco) and
incubated at room temperature (23-25.degree. C.) for 5 min. The two
separate dilutions were mixed and incubated at room temperature
(23-25.degree. C.) for 10 min. The DNA-Expifectamin.TM. 293 mix was
added directly to 1 mL EXPI293F cell culture. At the time of
transfection the cell density of the EXPI293F culture should be
2.8-3.2.times.10E6 cells/mL. The transfected cell cultures were
incubated in an orbital shaker incubator at 36.5.degree. C., 8% CO2
and 85-125 rpm. 18 hrs post transfection; 5 uL Expifectamin.TM. 293
Transfection Enhancer 1 and 50 uL Expifectamin.TM. 293 Transfection
Enhancer 2 were added per 1-mL culture. 5 days post transfection;
the cell culture supernatants were harvested by centrifugation,
followed by filtration through a 0.22 .mu.m PES filter unit
(Corning).
Purification
The fusion proteins were captured on a Gigacap column (ion
exchange) at pH 8 (neutral pH) and eluted with an increase in salt
(sodium sulphate) concentration using a stepwise gradient. The
eluted protein was either concentrated on Amicon Ultra centrifugal
filters with a MWCO of 10 kDa or not, depending on the
concentration in the capture pool. The analogue was finally
purified on a HiLoad Superdex200 16/60 or 26/60 prep grade column
using a PBS buffer.
General Methods of Detection and Characterisation
MS Analysis
Intact mass of the purified combined fusion protein was analysed
using Thermo-Dionex Ultimate3000.TM. HPLC (Thermo Fisher
Scientific) coupled to a Maxis Impact.TM. ESI-Q-OTOF mass
spectrometer (Bruker Daltonics). Solvents were A: Water with 0.1%
Formic Acid (v/v) and B: Acetonitrile with 0.08% Formic acid (v/v).
The sample was desalted online on a Waters Acquity.TM. BEH300 C4
1.7 .mu.m 1.0.times.100 mm column (Waters) for 2 minutes in 10% B,
0.2 ml/min and eluted by a 8 minute linear gradient from 10% B to
90% B solvent at 0.2 ml/min.
Absorbance at 215 nm (Abs215) and m/z spectra in the range m/z 300
to 3000 were recorded. Obtained data was analysed using the
DataAnalysis 4.1 software (Bruker Daltonics). Averaged m/z spectra
were deconvoluted using Maximum Entropy deconvolution.
Peptide mass mapping was performed to verify correct linker
sequences and was done using methods know to persons skilled in the
art. In short, purified proteins were subjected to tryptic
digestion using a method adopted from "In solution tryptic digest
and guanidation kit", Pierce product nr. 89895. Peptide mass
mapping to allow identification and verification of the correct
linker sequence in the fusion proteins was done using the Data
analysis software (Bruker Daltonics) to extract experimental
determined masses of peptides and the Biotools software (Biotools)
for matching experimental masses against the calculated masses
derived from the expected fusion protein sequences according to the
manufacturer's instructions. In general, variable modifications
were set to "Oxidation (M)" and "Carbamidomethyl (C)" and the mass
tolerance was set to 20 ppm and MS/MS tolerance to 50 mmu.
Chemiluminescent Nitrogen Detection (CLND) coupled to a standard
HPLC was used to determine the protein concentration essentially as
described elsewhere (eg. Bizanek, R.; Manes, 3. D.; Fujinari, E. M.
Chemiluminescent nitrogen detection as a new technique for purity
assessment of synthetic peptides separated by reversed-phase HPLC.
Pept. Res. 1996, 9 (1), 40-44).
Example 1: Expression and Purification of the Compounds of the
Invention
The different plasmids encoding the fusion protein variants
depicted in Table 1 were designed with differences in the linker
sequence between the human serum albumin part and the MIC-1
part.
TABLE-US-00001 TABLE 1 List of HSA MIC-1 fusion proteins. Compounds
referred to in the table has human serum albumin or a human serum
albumin variant in the N-terminal, a linker sequence as indicated
with an amino acid sequence and wild type human MIC-1 or a MIC-1
functional variant in the C-terminal (see also FIG. 1). An
N-terminal His6 tag (SEQ ID NO: 3) was included for some constructs
to facilitate IMAC purification. Peptide Human General map serum
expression (sequence N-terminal albumin Linker MIC-1 method
coverage, Compound His-tag (HSA) sequence Protein used (%) 1 SEQ ID
SEQ ID No linker SEQ ID NO: 1 2 53.8 NO: 3 NO: 2 2 SEQ ID SEQ ID
SEQ ID NO: 4 SEQ ID NO: 1 2 68.3 NO: 3 NO: 2 3 SEQ ID SEQ ID SEQ ID
NO: 5 SEQ ID NO: 1 2 82.8 NO: 3 NO: 2 4 SEQ ID SEQ ID SEQ ID NO: 6
SEQ ID NO: 1 2 32.0 NO: 3 NO: 2 5 SEQ ID SEQ ID SEQ ID NO: 10 SEQ
ID NO: 1 2 60.5 NO: 3 NO: 2 6 SEQ ID SEQ ID SEQ ID NO: 8 SEQ ID NO:
1 2 70.0 NO: 3 NO: 2 7 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 1 2
53.6 NO: 3 NO: 2 8 SEQ ID SEQ ID SEQ ID NO: 7 SEQ ID NO: 1 2 62.9
NO: 3 NO: 2 9 SEQ ID SEQ ID SEQ ID NO: 11 SEQ ID NO: 1 2 69.5 NO: 3
NO: 2 10 SEQ ID SEQ ID SEQ ID NO: 12 SEQ ID NO: 1 2 66.3 NO: 3 NO:
2 11 SEQ ID SEQ ID SEQ ID NO: 13 SEQ ID NO: 1 2 71.6 NO: 3 NO: 2 12
SEQ ID SEQ ID SEQ ID NO: 24 SEQ ID NO: 1 2 49.4 NO: 3 NO: 2 13 SEQ
ID SEQ ID SEQ ID NO: 25 SEQ ID NO: 1 2 62.5 NO: 3 NO: 2 14 SEQ ID
SEQ ID SEQ ID NO: 26 SEQ ID NO: 1 2 64.1 NO: 3 NO: 2 15 SEQ ID SEQ
ID SEQ ID NO: 27 SEQ ID NO: 1 2 75.5 NO: 3 NO: 2 16 SEQ ID SEQ ID
SEQ ID NO: 28 SEQ ID NO: 1 2 64.8 NO: 3 NO: 2 17 SEQ ID SEQ ID SEQ
ID NO: 29 SEQ ID NO: 1 2 31.0 NO: 3 NO: 2 18 SEQ ID SEQ ID SEQ ID
NO: 30 SEQ ID NO: 1 2 73.1 NO: 3 NO: 2 19 SEQ ID SEQ ID SEQ ID NO:
31 SEQ ID NO: 1 2 84.7 NO: 3 NO: 2 20 SEQ ID SEQ ID SEQ ID NO: 32
SEQ ID NO: 1 2 78.3 NO: 3 NO: 2 21 SEQ ID SEQ ID SEQ ID NO: 33 SEQ
ID NO: 1 2 74.0 NO: 3 NO: 2 22 SEQ ID SEQ ID SEQ ID NO: 34 SEQ ID
NO: 1 2 81.8 NO: 3 NO: 2 23 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO:
1 3 75.1 tag NO: 2 24 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 1 3
79.3 tag NO: 23 25 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 14 3 71.0
tag NO: 23 26 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 15 3 73.8 tag
NO: 23 27 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 16 2 87.0 NO: 3 NO:
23 28 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 17 2 86.5 NO: 3 NO: 23
29 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 18 2 84.3 NO: 3 NO: 23 30
SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 19 2 82.2 NO: 3 NO: 23 31 SEQ
ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 20 2 91.1 NO: 3 NO: 23 32 SEQ ID
SEQ ID SEQ ID NO: 9 SEQ ID NO: 21 2 83.6 NO: 3 NO: 23 33 SEQ ID SEQ
ID SEQ ID NO: 35 SEQ ID NO: 1 2 62.6 NO: 3 NO: 2 34 SEQ ID SEQ ID
SEQ ID NO: 36 SEQ ID NO: 1 2 75.1 NO: 3 NO: 2 35 SEQ ID SEQ ID SEQ
ID NO: 37 SEQ ID NO: 1 2 79.6 NO: 3 NO: 2 36 SEQ ID SEQ ID SEQ ID
NO: 38 SEQ ID NO: 1 2 74.1 NO: 3 NO: 2 37 SEQ ID SEQ ID SEQ ID NO:
9 SEQ ID NO: 22 2 76.4 NO: 3 NO: 23
Some fusion proteins comprise wild type human MIC-1 (SEQ ID NO:1),
others MIC-1 variants (SEQ ID NO:14-SEQ ID NO:23). Some fusion
proteins comprises human wild type human serum albumin (SEQ ID
NO:2), others HSA C34A, a human serum albumin variant wherein the
cysteine residue at position 34 of the wild type human serum
albumin amino acid sequence has been replaced with alanine (SEQ ID
NO:23). Plasmids were generated by well-known recombinant DNA
technology methods (obtained from GenScript Inc). Constructs were
designed with or without a N-terminal His tag sequence (SEQ ID
NO:3). Constructs with His tag allows direct purification using
immobilized affinity chromatography (IMAC), whereas other means of
purification was used for purification of non-His tagged
constructs. Since the His-tag is placed in the very N-terminal of
human serum albumin it does not affect neither the efficacy of the
MIC-1 fusion proteins, nor the binding of human serum albumin to Fc
Neonatal Receptor and the half-life extending effect of human serum
albumin, when used as a fusion partner. The linker sequence is
given in Table 2.
TABLE-US-00002 TABLE 2 List of peptide linkers with corresponding
SEQ ID NO and amino acid sequence. SEQ ID NO Linker sequence SEQ ID
NO: 4 EAAEAAE SEQ ID NO: 5 EEEAEEEAEEEAEEEAEEE SEQ ID NO: 6
GGSSSGSGGSGGSGSGGSGGSGS SEQ ID NO: 7 DDADDADDADDADDADDAD SEQ ID NO:
8 KAAKAAKAAKAAKAAKAAK SEQ ID NO: 9 GGSSEAAEAAEAAEAAEAAEAAE SEQ ID
NO: 10 DAADAADAADAADAADAAD SEQ ID NO: 11 EAAEAAEAAEAAEAAEAAE SEQ ID
NO: 12 EAAEAAEAAEAAEAAEAAEAAEAAEAAEAAE SEQ ID NO: 13
GGSSEAAEAAEAAEAAEAAEAAEAAEAAEAAEAAE SEQ ID NO: 24 AAEGEEEAE SEQ ID
NO: 25 GGSSSGS SEQ ID NO: 26 PTPTPTP SEQ ID NO: 27
GGSSEEEAEEEAEEEAEEEAEEE SEQ ID NO: 28
GGSSSGSGGSGGSGSGGSGGSGSGSGGSGGS SEQ ID N0: 29
GGSSPTPTPTPTPTPTPTPTPTP SEQ ID NO: 30
PTPTPTPTPTPTPTPTPTPTPTPTPTPTPTP SEQ ID NO: 31
QAAAQAAAQAAAQAAAQAAAQAAAQ SEQ ID N0: 32 QAAQAAQAAQAAQAAQAAQ SEQ ID
NO: 33 EAAAEAAAEAAAEAAAEAAAE SEQ ID NO: 34 DAAADAAADAAADAAADAAAD
SEQ ID NO: 35 GGSSEAAAEAAAEAAAEAAAEAAAE SEQ ID NO: 36
EAAAEAAAEAAAEAAAEAAAEAAAE SEQ ID NO: 37 DAAADAAADAAADAAADAAADAAAD
SEQ ID NO: 38 GGSSEAAAEAAAEAAAEAAAEAAAEAAAE
TABLE-US-00003 TABLE 3 List of MIC-1 variants with corresponding
SEQ ID NO. SEQ ID NO MIC-1 variants SEQ ID NO: 1 Wild type human
MIC-1 (hMIC-1) SEQ ID NO: 14 N3S hMIC-1 SEQ ID NO: 15 R2A, N3E
hMIC-1 SEQ ID NO: 16 N3E hMIC-1 SEQ ID NO: 17 N3A hMIC-1 SEQ ID NO:
18 N3P hMIC-1 SEQ ID NO: 19 N3T hMIC-1 SEQ ID NO: 20 N3G hMIC-1 SEQ
ID NO: 21 N3Q hMIC-1 SEQ ID NO: 22 N3D hMIC-1
TABLE-US-00004 TABLE 4 List of human serum albumin (HSA) variants
with corresponding SEQ ID NO. SEQ ID NO Human serum albumin
variants SEQ ID NO: 2 Wild type HSA SEQ ID NO: 23 C34A HSA
As a representative example, large scale production of Compound no.
7 was performed by transient expression in Expi293F cells as
described in materials and methods section. Briefly, 200 .mu.g
plasmid DNA was added to 10 ml of Opti-MEM.RTM. transfection medium
and 540 .mu.l ExpiFectamine.TM. 293 reagent was added to 10 ml of
Opti-MEM.RTM. transfection medium. The two solutions were combined
to form a transfection mix. After 20 minutes incubation, the
transfection mix was added to 200 ml of expi293F cell culture with
a cell density of 3.times.106 cells/ml. 18 hours after
transfection, the cultures were fed with 1 ml of enhancer 1 and 10
ml of enhancer 2. Approximately 90 hours after transfection the
culture was harvested by centrifugation at 4000 g for 10 minutes.
The clarified medium was sterile filtered through a 0.22 uM filter
before purification.
To examine the in vivo effect of fusing a human serum albumin
molecule to the N-terminus of the MIC-1 protein by variable linkers
the expressed molecule were purified using the method described
above. Compound no. 7 was successfully purified using automated
immobilized metal ion chromatography coupled to size exclusion. Two
major peaks within the total volume of the SEC column were
fractioned and analysed. The first peak eluted at the void of the
column and non-reducing SDS-PAGE confirmed the aggregated state of
the eluted protein. The main peak partially overlapped with the
aggregate peak. Therefore, not all fractions representing the
entire main peak were included in the pool. Non-reducing SDS-PAGE
of the pooled fractions resulted in a single band which migrated as
a .about.120 kDa protein. To verify the dimeric structure of the
molecule, intact mass spectrometry (MS) was performed.
Deconvolution of the averaged mass spectra resulted in the average
mass 164140 Da. The calculated molecular weight of Compound no. 7
is 163820 Da. Thus, intact MS analysis shows that the purified
molecule is in its dimeric form, but potentially carries several
post translational modifications (e.g. oxidations, deamidations
etc.). To further characterise the constructs, peptide mass mapping
strategies were deployed for characterisation. HSA-MIC-1 fusion
proteins expressed in the mammalian host cells, produced varying
degree of Cys34 cysteinylation as described previously (Kleinova A,
et al., Rapid Commun. Mass Spectrom, 2005; 19: 2965-2973.). In
addition, it was found that other causes of heterogeniety was
linked to the Asn in position 3 of the MIC-1 sequence, which was
found highly labile, since it readily deamidated to Asp or
isoAsp.
Pharmacological Methods
Example 2: Effect of Fusions Proteins of the Invention on Food
Intake in Lean Sprague Dawley Rats
The purpose of this example is to test the efficacy of the
compounds in vivo. The in vivo efficacy of the compounds of the
invention was measured in 250 g-300 g male non-obese Sprague Dawley
rats. Animals were injected once with a dose of 4 nmol/kg body
weight. Compounds were administrate subcutaneously (1 ml/kg) in a
physiological isotonic phosphate buffered saline (PBS) solution
(137 mM NaCL; 2.7 mM KCl; 10 mM Na.sub.2HPO.sub.4; 1.8 mM
KH.sub.2PO.sub.4). In some cases the buffered saline solution also
contained 500 ppm of polysorbate 80. Wild-type human MIC-1 was
included as a reference compound and was injected once daily during
the study with a dose of 8 nmol/kg body weight. Wild-type hMIC-1
was administered subcutaneously (1 ml/kg) in an acidic isotonic
buffered solution (pH 4.0; 5 mM acetate, 2.25% glycerol, 70 ppm
polysorbate 20).
Changes in food intake were measured either by an automatic food
monitoring system (BioDAQ or HM-2) or by measuring the reduction in
food pellets in the cage feeding tray manually over a 24 hr period
of time. Animals were single housed in the BioDAQ system and housed
3 per cage in the HM-2 system. Animals were in the latter system
chip-marked prior study start in order for the HM-2 system to
collect individual measures of food intake. Each compound was
tested in n=4-8 animals in one or more experiments. Animals were
acclimatized for at least for 7 days in the experimental set up
prior to study start. Collected data are expressed as daily food
intake (24 hour food intake) measured from the onset of each daily
12 hour dark phase to the following dark phase. Daily changes in
food intake in response to administered compound were in most
studies calculated by subtracting the average daily food intake of
the treatment group from the average daily food intake of the
vehicle group. In a few studies daily changes in food intake in
response to administered compound were calculated by subtracting
the daily average food intake during the intervention from the
average daily food intake of the day prior to study start. Changes
were considered significant if p<0.1 using a student's t-test
(two-tailed).
Several amino acid linkers between the human serum albumin part and
the MIC-1 part were explored. The linkers were characterised by
having different lengths, charges or structural motifs (eg.
Pro-rich linkers, linkers with predicted alpha-helical propensities
comprising Glu/Asp and Ala or typical Gly/Ser containing linkers
conferring linker flexibility). The linker variants were evaluated
and compared on basis of max efficacy, duration of biological
effect and accumulated efficacy as described above.
The inventors surprisingly found that the absence of a linker or
peptide linkers with a size below 10 amino acids between human
serum albumin and MIC-1 resulted in compounds with limited or no
significant biological efficacy (compounds 1, 2, 12, 13 and 14
(table 5)).
In contrast, linkers with a size of 10 or more amino acids
positively influenced the biological efficacy of the fusion
protein. In the present invention it was also found that variation
in the linker amino acid composition and sequence also
significantly influenced the biological efficacy of the HSA MIC-1
fusion proteins. The inventors surprisingly found that a specific
combination of medium sized linkers of about 20 amino acids
comprising repeats of an acidic residue (Glu or Asp) followed by at
least two non-polar residues such as Ala resulted in increased
biological efficacy of the HSA-MIC-1 fusion protein when compared
to flexible linkers of identical size comprising Gly and Ser
residues, which are normally used as linkers for separating domains
of fusion proteins (Table 3, Compound no. 7 or 9 compared with
Compound no. 4).
TABLE-US-00005 TABLE 5 Effect of a single dose (4 nmol/kg) of
comparative HSA-MIC-1 fusion proteins on daily food intake in lean
SD rats. Data are expressed in 3 ways, 1) maximum efficacy which is
the greatest significant (p < 0.10) reduction in 24 hours food
intake recorded over the study period, 2) Accumulated efficacy
which is the sum of significant (p < 0.10) reductions in 24
hours food intake compared with vehicle and 3) Duration of effect
which is the number of days with a significant (p < 0.1)
reductions in food intake compared with vehicle. Wild-type human
MIC-1 is included for comparison and was administered once daily
for 7 days (8 nmol/kg). Length of linker (number of Maximum
Accumulated Duration of Compound amino acids) efficacy efficacy
effect wt MIC-1 0 37 n/a 1 (SEQ ID NO: 1) 1 0 0 0 0 2 7 11 11 1 12
9 11 8 1 13 7 -12 -12 1 14 7 15 1 2 8 19 23 66 3 3 19 30 74 4 15 23
20 38 2 4 23 28 72 3 16 31 22 56 3 6 19 18 34 2 17 23 30 78 3 18 31
23 23 1 19 25 20 51 3 20 19 30 83 3
TABLE-US-00006 TABLE 6 Effect of a single dose (4 nmol/kg) of
HSA-MIC-1 fusion proteins of the invention having varying linker
length on daily food intake in lean SD rats. Data are expressed in
3 ways, 1) maximum efficacy which is the greatest significant (p
< 0.10) reduction in 24 hours food intake recorded over the
study period, 2) Accumulated efficacy which is the sum of
significant (p < 0.10) reductions in 24 hours food intake
compared with vehicle and 3) Duration of effect which is the number
of days with a significant (p < 0.1) reductions in food intake
compared with vehicle. Wild-type human MIC-1 is included for
comparison and was administered once daily for 7 days (8 nmol/kg).
Length of linker (number of Maximum Accumulated Duration of
Compound amino acids) efficacy efficacy effect wt MIC-1 0 37 n/a 1
(SEQ ID NO: 1) 5 19 42 183 6 g 19 35 86 4 21 21 33 80 3 22 21 39
183 6 7 23 39 167 6 23 23 31 98 4 33 25 37 154 5 34 25 31 135 5 35
25 38 149 5 36 29 39 169 6 10 31 31 91 4 11 35 25 60 3
More in particular, the inventors surprisingly found that medium
sized linkers of about 10-35 amino acids comprising repeats of an
acidic residue (Glu or Asp) followed by at least two non-polar
residues such as Ala showed favorable biological efficacy of the
HSA-MIC-1 fusion protein, when compared to flexible linkers of
identical size comprising Gly and Ser residues or rigid Pro
containing linkers (compound 7 (table 6) compared with compounds 4
and 17 (table 5)). Similar observations were done for longer
linkers above 30 aa (compound 10 (table 6) compared with compound
16 or 18 (table 5)). Substitutions of Ala with acidic residues in
each repeat negatively affected the maximum efficacy and/or
accumulated efficacy (e.g. compound 5 (table 6) compared to
compound 8 (table 5)). Surprisingly, it was found that substitution
of the acidic residues of linkers containing repeats of Glu-Ala-Ala
or Asp-Ala-Ala with a basic Lys residue resulted in a clear
decrease in biological efficacy and accumulated food intake
demonstrating that difference in efficacy can result from small
changes in the linker sequence (Compound no. 5 and 9 (table 6)
compared to compound 6 (table 5)).
Thus, the present invention demonstrates that HSA MIC-1 fusion
proteins with certain linkers results in higher maximum efficacy
and accumulated efficacy as well as longer duration of the fusion
protein.
TABLE-US-00007 TABLE 7 Effect of a single dose (4 nmol/kg) of
HSA-MIC-1 fusion proteins of the invention all having a linker of
SEQ ID NO: 9, and comprising a MIC-1 variant and/or human serum
albumin variant on daily food intake in lean SD rats. Data are
expressed in 3 ways, 1) maximum efficacy which is the greatest
significant (p < 0.10) reduction in 24 hours food intake
recorded over the study period, 2) Accumulated efficacy which is
the sum of significant (p < 0.10) reductions in 24 hours food
intake compared with vehicle and 3) Duration of effect which is the
number of days with a significant (p < 0.1) reductions in food
intake compared with vehicle. Wild-type hMIC-1 is included for
comparison and was administered once daily for 7 days (8 nmol/kg).
Max- Accum- Dura- MIC-1/ imum ulated tion of Compound HSA variant
MIC-1 variant efficacy efficacy effect wt MIC-1 -- -- 37 n/a 1 (SEQ
ID NO: 1) 24 SEQ ID NO: 23 SEQ ID NO: 1 29 69 3 25 SEQ ID NO: 23
SEQ ID NO: 14 38 127 5 26 SEQ ID NO: 23 SEQ ID NO: 15 30 114 5 27
SEQ ID NO: 23 SEQ ID NO: 16 31 87 3 28 SEQ ID NO: 23 SEQ ID NO: 17
31 89 4 29 SEQ ID NO: 23 SEQ ID NO: 18 38 78 3 30 SEQ ID NO: 23 SEQ
ID NO: 19 22 54 3 31 SEQ ID NO: 23 SEQ ID NO: 20 30 74 3 32 SEQ ID
NO: 23 SEQ ID NO: 21 34 113 4 37 SEQ ID NO: 23 SEQ ID NO: 22 44 173
5
HSA MIC-1 fusions proteins, all with a linker of SEQ ID NO:9, and
comprising the MIC-1 variants and/or the human serum albumin
variants of tables 3 and 4, respectively, were prepared and tested
to investigate if these changes in the MIC-1 part and/or the human
serum albumin part had an effect on the efficacy of the fusion
proteins. As can be seen in table 7, all the fusions proteins were
found to significantly reduce food intake for 3-5 days in response
to a single injection of 4 nmol/kg.
Example 3: Effect of Fusions Proteins of the Invention on Food
Intake in DIO Sprague Dawley Rats
DIO rats were used to further study compounds tested in lean rats.
Obesity was induced by placing eight-week-old animals on a special
research diet (Research Diets, D12451) where 45% of the energy
content is derived from fat. Animals typically reached a body
weight of 500-600 g before study initiation. Animals were injected
once with a dose of 4 nmol/kg body weight. Compounds were
administered subcutaneously (1 ml/kg) in a physiological isotonic
phosphate buffered saline (PBS) solution (137 mM NaCL; 2.7 mM KCl;
10 mM Na.sub.2HPO.sub.4; 1.8 mM KH.sub.2PO.sub.4). In some cases
the buffered saline solution also contained 500 ppm of polysorbate
80.
Changes in food intake were measured by an automatic food
monitoring system (BioDAQ or HM-2). Animals were single housed in
the BioDAQ system and housed 3 per cage in the in the HM-2 system.
Animals were in the latter system chip-marked prior to study start
in order for the HM-2 system to collect individual measures of food
intake. Each compound was tested in n=4-8 animals in one or more
experiments. Animals were acclimatized for at least 7 days in the
experimental set up prior to study start. Collected food intake
data are expressed as daily food intake (24 hour food intake)
measure from the onset of each daily 12 hour dark phase to the
following dark phase. Daily changes in food intake in response to
administered compound were calculated by subtracting the average
daily food intake of the treatment group from the average daily
food intake of the vehicle group. Changes were considered
significant if p<0.1 using a student's t-test (two-tailed).
The HSA MIC-1 fusion proteins tested all displayed good efficacy in
DIO rats. When comparing compound 7 and 23, it is apparent that the
His-tag (SEQ ID NO:3) does not affect the efficacy or duration of
effect of the fusion proteins.
TABLE-US-00008 TABLE 8 Effect of a single dose (4 nmol/kg) of
HSA-MIC-1 analogues on body weight and daily food intake in obese
SD rats. Data are expressed in 4 ways, 1) maximum efficacy which is
the greatest significant (p < 0.10) reduction in 24 hours food
intake recorded over the study period, 2) Accumulated efficacy
which is the sum of significant (p < 0.10) reductions in 24
hours food intake compared with vehicle and 3) Duration of effect
which is the number of days with a significant (p < 0.1)
reductions in food intake compared with vehicle, 4) Differences in
body weight at day 7 compared to the vehicle group. Maximum
Accumulated Duration of % body weight Compound efficacy efficacy
effect difference 5 61 309 6 -6.7 7 68 369 6 -8.9 9 56 331 7 -7.1
23 70 355 7 -8.8 24 62 331 6 -10 26 64 385 7 -9.6
Example 4: Pharmacokinetic Evaluation of MIC-1 Compounds in Lean
Sprague Dawley Rats
The purpose of this study is to determine the half-life in vivo of
the HSA MIC-1 fusion proteins after intravenous administration to
lean Sprague Dawley rats, i.e. the prolongation of their time in
the blood circulation and thereby their time of action. This is
done in a pharmacokinetic (PK) study, where the terminal half-life
of the fusion protein in question is determined. By terminal
half-life is generally meant the period of time it takes to halve a
certain plasma concentration, measured after the initial
distribution phase.
The in vivo half-life was measured in 300 g-500 g lean SD rats by
injecting the compound into the tail vein followed by collection of
blood plasma samples at various time points for exposure analysis.
Compounds (0.5 nmol/kg body weight) were administered intravenously
(1 ml/kg) in a physiologically isotonic phosphate buffered saline
(PBS) solution (140 mM NaCL; 2.7 mM KCl; 8.05 mM Na.sub.2HPO.sub.4;
1.96 mM KH.sub.2PO.sub.4, 500 ppm polysorbate 80). Blood samples
were collected from the tongue at time -30, 30, 60, 240 and 420
minutes and 24, 30, 48, 72, 96, 120, 168, 216, 264 and 360/384
hours. 200 .mu.l of blood was collected into EDTA tubes and stored
on ice for up to 20 minutes. Plasma samples were generated by
centrifuging blood samples for 5 minutes at 10000 G at 4.degree. C.
The sample was subsequent pipetted into Micronic tubes on dry ice,
and kept at -20.degree. C. until analysed for plasma concentration
of the respective MIC-1 compound using LOCI or a similar antibody
based assay such as ELISA. The individual plasma concentration-time
profiles were analysed by a non-compartmental model in Phoenix v.
6.2 or 6.3 software (Pharsight Inc., Mountain View, Calif., USA),
and the resulting terminal half-lives determined.
TABLE-US-00009 TABLE 9 Pharmacokinetic profile of MIC-1 compounds
in lean SD rats (0.5 nmol/kg) with intravenous tail vein dosing.
Data are expressed as the half-life (T1/2). Compound intravenous
T1/2 (hours) wt hMIC-1 (SEQ ID NO: 1) 1.9 1 38 2 32 3 27 4 29 5 27
6 27 7 36 8 19 9 37 10 2 13 2 14 41 16 3 18 45 20 35 21 39 23 46 24
42 25 38 26 49 33 36 35 31 36 37
A correlation between the length of the linker (i.e--the number of
amino acids in the linker) and the T1/2 in lean rat was analysed
using a Pearson correlation analysis. The Spearman correlation
coefficient was -0.0365 suggesting no significant linear
relationship between the linker length and T1/2. An implication of
this analysis is that the biological efficacy of the fusion
proteins is not a function of the in vivo half-life of the fusion
proteins.
While certain features of the invention have been illustrated and
described herein, many modifications, substitutions, changes, and
equivalents will now occur to those of ordinary skill in the art.
It is, therefore, to be understood that the appended claims are
intended to cover all such modifications and changes as fall within
the true spirit of the invention.
SEQUENCE LISTINGS
1
401112PRTHOMO SAPIENS 1Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro
Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala Ser Leu Glu
Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro Arg Glu Val
Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser Gln Phe Arg
Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60 Leu His Arg
Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65 70 75 80 Ala
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90
95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110 2585PRTHOMO SAPIENS 2Asp Ala His Lys Ser Glu Val Ala
His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65
70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310
315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435
440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu
His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala
Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555
560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585
311PRTARTIFICIALHis-tag 3His His His His His His Gly Gly Gly Ser
Ser 1 5 10 47PRTARTIFICIALpeptide linker 4Glu Ala Ala Glu Ala Ala
Glu 1 5 519PRTARTIFICIALpeptide linker 5Glu Glu Glu Ala Glu Glu Glu
Ala Glu Glu Glu Ala Glu Glu Glu Ala 1 5 10 15 Glu Glu Glu
623PRTARTIFICIALpeptide linker 6Gly Gly Ser Ser Ser Gly Ser Gly Gly
Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Ser
20 719PRTARTIFICIALpeptide linker 7Asp Asp Ala Asp Asp Ala Asp Asp
Ala Asp Asp Ala Asp Asp Ala Asp 1 5 10 15 Asp Ala Asp
819PRTARTIFICIALpeptide linker 8Lys Ala Ala Lys Ala Ala Lys Ala Ala
Lys Ala Ala Lys Ala Ala Lys 1 5 10 15 Ala Ala Lys
923PRTARTIFICIALpeptide linker 9Gly Gly Ser Ser Glu Ala Ala Glu Ala
Ala Glu Ala Ala Glu Ala Ala 1 5 10 15 Glu Ala Ala Glu Ala Ala Glu
20 1019PRTARTIFICIALpeptide linker 10Asp Ala Ala Asp Ala Ala Asp
Ala Ala Asp Ala Ala Asp Ala Ala Asp 1 5 10 15 Ala Ala Asp
1119PRTARTIFICIALpeptide linker 11Glu Ala Ala Glu Ala Ala Glu Ala
Ala Glu Ala Ala Glu Ala Ala Glu 1 5 10 15 Ala Ala Glu
1231PRTARTIFICIALpeptide linker 12Glu Ala Ala Glu Ala Ala Glu Ala
Ala Glu Ala Ala Glu Ala Ala Glu 1 5 10 15 Ala Ala Glu Ala Ala Glu
Ala Ala Glu Ala Ala Glu Ala Ala Glu 20 25 30
1335PRTARTIFICIALpeptide linker 13Gly Gly Ser Ser Glu Ala Ala Glu
Ala Ala Glu Ala Ala Glu Ala Ala 1 5 10 15 Glu Ala Ala Glu Ala Ala
Glu Ala Ala Glu Ala Ala Glu Ala Ala Glu 20 25 30 Ala Ala Glu 35
14112PRTARTIFICIALMIC-1 analogue 14Ala Arg Ser Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65
70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 15112PRTARTIFICIALMIC-1 analogue 15Ala
Ala Glu Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10
15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln
Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro
Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu
Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110
16112PRTARTIFICIALMIC-1 analogue 16Ala Arg Glu Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65
70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 17112PRTARTIFICIALMIC-1 analogue 17Ala
Arg Ala Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10
15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln
Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro
Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu
Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110
18112PRTARTIFICIALMIC-1 analogue 18Ala Arg Pro Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65
70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 19112PRTARTIFICIALMIC-1 analogue 19Ala
Arg Thr Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10
15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln
Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro
Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu
Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110
20112PRTARTIFICIALMIC-1 analogue 20Ala Arg Gly Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65
70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 21112PRTARTIFICIALMIC-1 analogue 21Ala
Arg Gln Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10
15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln
Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro
Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu
Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110
22112PRTARTIFICIALMIC-1 analogue 22Ala Arg Asp Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65
70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 23585PRTARTIFICIALvariant of human
serum albumin 23Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30 Gln Ala Pro Phe Glu Asp His Val Lys
Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala
Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu
Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr
Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105
110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys
Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe
Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230
235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325
330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys
Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp
Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro
Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu
Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala
Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr
Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly
Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450
455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys
His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met
Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570
575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585
249PRTARTIFICIALpeptide linker 24Ala Ala Glu Gly Glu Glu Glu Ala
Glu 1 5 257PRTARTIFICIALpeptide linker 25Gly Gly Ser Ser Ser Gly
Ser 1 5 267PRTARTIFICIALpeptide linker 26Pro Thr Pro Thr Pro Thr
Pro 1 5 2723PRTARTIFICIALpeptide linker 27Gly Gly Ser Ser Glu Glu
Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala 1 5 10 15 Glu Glu Glu Ala
Glu Glu Glu 20 2831PRTARTIFICIALpeptide linker 28Gly Gly Ser Ser
Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 Gly Ser
Gly Gly Ser Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30
2923PRTARTIFICIALpeptide linker 29Gly Gly Ser Ser Pro Thr Pro Thr
Pro Thr Pro Thr Pro Thr Pro Thr 1 5 10 15 Pro Thr Pro Thr Pro Thr
Pro 20 3031PRTARTIFICIALpeptide linker 30Pro Thr Pro Thr Pro Thr
Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr 1 5 10 15 Pro Thr Pro Thr
Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro 20 25 30
3125PRTARTIFICIALpeptide linker 31Gln Ala Ala Ala Gln Ala Ala Ala
Gln Ala Ala Ala Gln Ala Ala Ala 1 5 10 15 Gln Ala Ala Ala Gln Ala
Ala Ala Gln 20 25 3219PRTARTIFICIALpeptide linker 32Gln Ala Ala Gln
Ala Ala Gln Ala Ala Gln Ala Ala Gln Ala Ala Gln 1 5 10 15 Ala Ala
Gln 3321PRTARTIFICIALpeptide linker 33Glu Ala Ala Ala Glu Ala Ala
Ala Glu Ala Ala Ala Glu Ala Ala Ala 1 5 10 15 Glu Ala Ala Ala Glu
20 3421PRTARTIFICIALpeptide linker 34Asp Ala Ala Ala Asp Ala Ala
Ala Asp Ala Ala Ala Asp Ala Ala Ala 1 5 10 15 Asp Ala Ala Ala Asp
20 3525PRTARTIFICIALpeptide linker 35Gly Gly Ser Ser Glu Ala Ala
Ala Glu Ala Ala Ala Glu Ala Ala Ala 1 5 10 15 Glu Ala Ala Ala Glu
Ala Ala Ala Glu 20 25 3625PRTARTIFICIALpeptide linker 36Glu Ala Ala
Ala Glu Ala Ala Ala Glu Ala Ala Ala Glu Ala Ala Ala 1 5 10 15 Glu
Ala Ala Ala Glu Ala Ala Ala Glu 20 25 3725PRTARTIFICIALpeptide
linker 37Asp Ala Ala Ala Asp Ala Ala Ala Asp Ala Ala Ala Asp Ala
Ala Ala 1 5 10 15 Asp Ala Ala Ala Asp Ala Ala Ala Asp 20 25
3829PRTARTIFICIALpeptide linker 38Gly Gly Ser Ser Glu Ala Ala Ala
Glu Ala Ala Ala Glu Ala Ala Ala 1 5 10 15 Glu Ala Ala Ala Glu Ala
Ala Ala Glu Ala Ala Ala Glu 20 25 3918PRTArtificial Sequencepeptide
linker 39Glu Ala Ala Glu Ala Ala Glu Ala Ala Glu Ala Ala Glu Ala
Ala Glu 1 5 10 15 Ala Ala 404PRTArtificial SequenceSynthetic 40Gly
Gly Ser Ser 1
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